Methods, kits, and systems for multiplexed detection of target molecules and uses thereof

ABSTRACT

Described herein are methods, compositions, kits and systems for multiplexed detection of target molecules from a sample. In some embodiments, the methods, compositions, kits and systems can be used to perform multiplexed protein analysis of a sample (e.g., a sample comprising a small number of cells or a single-cell sample). In some embodiments, the same sample subjected to a multiplexed protein analysis using the methods, compositions, kits and systems described herein can also be subjected to a nucleic acid (e.g., RNAs, microRNAs, and/or DNA) analysis, thereby creating an integrated expression profiling from a limited amount of sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/897,025 filed Dec. 9, 2015, which application is a 35 U.S.C. § 371National Phase Entry Application of International Application No.PCT/US2014/040731 filed Jun. 3, 2014, which designates the U.S., andclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/834,111 filed Jun. 12, 2013, U.S. ProvisionalApplication No. 61/912,054 filed Dec. 5, 2013, U.S. ProvisionalApplication No. 61/972,940 filed Mar. 31, 2014, the contents of each ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 4, 2019, isnamed 030258-078413-PCT_SL, and is 34,864 bytes in size.

TECHNICAL FIELD

The present invention relates to methods, kits, and systems fordetection of a plurality of target molecules in a sample. The methods,kits, and systems described herein can be used in diagnostic,prognostic, quality control and screening applications.

BACKGROUND

An increasing number of clinical trials, e.g., cancer trials, requirepatient samples, e.g., tissue biopsies, to measure individual drugresponse markers [1]. For example, surgically harvested tissues areoften used to collect data at two ends of the cellular spectrum: (i)genomic analyses that reveal driver oncogenes and specific mutations [2]and (ii) protein analyses of selected biomarkers intended to monitorcellular responses [3, 4]. Ideally, clinical samples are collectedserially to monitor change in expression levels of key proteins. Thisraises many challenges, notably risk of morbidity with repeat corebiopsies, increased cost, and logistical limitations. Alternative samplecollection methods include fine-needle aspirates (FNAs), “liquidbiopsies” of circulating tumor cells, or analysis of scant cells presentin other easily harvested fluids. However, these samples have much lowercell numbers than biopsies, thereby limiting the number of proteins thatcan be analyzed.

After tissues have been sampled, selecting ubiquitous biomarkers can bedifficult because of heterogeneity and dynamic network changes.Typically, small-molecule drugs influence more than one target proteins,whereas numerous proteins modulate downstream specific drug actions,trigger alternative molecular pathways, and induce tumor cell death orresistance [5]. The current tools to profile these key proteins in scantclinical samples are limited; standard practice encompassesimmunocytology, which often precludes broad protein analysis because ofinsufficient sample within FNAs or liquid biopsies [6]. Thus, the numberof markers is often limited (<10) and requires time-consuming analysesof tissue sections. Proteomic analyses by mass spectrometry remaintechnically challenging for single cells and phosphoproteomic detectionand are costly for routine clinical purposes [7]. In research settings,multiplexed flow cytometry and mass cytometry have been used to examinean expanded set of markers (10 to 45) using single-cell populations.However, multiplexed flow cytometry often encounters limits in theamount of markers it can measure because of spectral overlap. Masscytometry vaporizes cells during sample preparation, resulting in sampleloss [8]. Accordingly, both of these existing methods do not enableisolating a rare cell of interest or performing concurrent geneticanalyses once samples are used for proteomic analyses.

Hence, there remains a need for compositions and methods forsimultaneous detection of a large number of target molecules from asample.

SUMMARY

Embodiments of various aspects described herein are, in part, based onthe development of a method that not only allows analysis of hundreds ofproteins from a limited amount of sample, e.g., minimally invasivefine-needle aspirates (FNAs), which contain much smaller numbers ofcells than core biopsies, but also preserves genetic material from thesame sample to enable simultaneous measurements of proteins and geneticmaterials (e.g., DNA, RNA, and microRNAs). In particular, the methodrelies on DNA-barcoded antibody sensing, where barcodes-single strandsof DNA- can be photocleaved and detected using fluorescent complementaryprobes without any amplification steps, and is referred to as anantibody barcoding with photocleavable DNA (ABCD) platform herein. Todemonstrate the capability of the ABCD platform, inventors isolatedcancer cells within the FNAs of patients and exposed these cells to amixture of about 90 DNA-barcoded antibodies, covering the hallmarkprocesses in cancer (for example, apoptosis and DNA damage). Theinventors discovered that the single-cell protein analysis of thepatients' FNAs showed high intratumor heterogeneity, indicating theability of the ABCD platform to perform protein profiling on rare singlecells, including, but not limited to circulating tumor cells. Further,the inventors discovered that patients who showed identicalhistopathology yet showed patient heterogeneity in proteomic profiling,indicating the ability of the ABCD platform to identify personalizedtargets for treatment. By profiling and clustering protein expression inpatients' samples, the inventors also showed use of the ABCD platform tomonitor and predict treatment response in patients receivingchemotherapy, e.g., kinase inhibitors. The protein analysis determinedby the ABCD platform is scalable and can be extended to detect othertarget molecules, e.g., metabolites and lipids. Accordingly, variousaspects described herein provide for methods, systems and kits fordetecting and/or quantifying a plurality of target molecules from asample, as well as their uses thereof in various applications, e.g.,diagnosis, prognosis, personalized treatment, and/or treatmentmonitoring.

In one aspect, provided herein is a method for detecting a plurality oftarget molecules in a sample. The method comprises (a) contacting asample with a composition comprising a plurality of target probes,wherein each target probe in the plurality comprises: (i) atarget-binding molecule that specifically binds to a target molecule inthe sample; (ii) an identification nucleotide sequence that identifiesthe target-binding molecule; and (iii) a cleavable linker between thetarget-binding molecule and the identification nucleotide sequence; (b)releasing the identification nucleotide sequences from the bound targetprobes; and (c) detecting signals from the released identificationnucleotide sequences, wherein the signals are distinguishable for theidentification nucleotide sequences, thereby identifying thecorresponding target-binding molecules and detecting a plurality oftarget molecules in the sample.

Stated another way, the method comprises: (a) forming a plurality ofcomplexes in a sample, each complex comprising a target molecule and atarget probe bound thereto, wherein the target probe comprises (i) atarget-binding molecule that specifically binds to the target moleculepresent in the sample; (ii) an identification nucleotide sequence thatidentifies the target-binding molecule; and (iii) a cleavable linkerbetween the target-binding molecule and the identification nucleotidesequence; (b) releasing the identification nucleotide sequences from thecomplex; and (c) detecting signals from the released identificationnucleotide sequences, wherein the signals are distinguishable for theidentification nucleotide sequences, thereby identifying thecorresponding target-binding molecules and detecting a plurality oftarget molecules in the sample. In some embodiments, the cleavablelinker is not pre-hybridized (e.g., by basepairing) to any portion ofthe identified nucleotide sequences.

In some embodiments, e.g., cell assay, each complex comprising a targetmolecule and a target probe bound thereto does not require two or moretarget probes of different kinds bound to the same target molecule,where each of the target probes binds to a different region of the sametarget molecule. For example, each complex does not require both a firsttarget probe binding to a first region of a target molecule, and asecond target probe binding to a second region of the same targetmolecule. Stated another way, in some embodiments, a single target probeas described herein binding to a target molecule is sufficient forenablement of the methods described herein. In these embodiments, themethod described herein does not require another target probe binding tothe same target molecule for attachment to a solid substrate (e.g., abead).

In some embodiments, the method can further comprise separating unboundtarget probes from target probes that are bound to the target moleculesin the sample.

The signals from the released identification nucleotide sequences can bedetected by various methods known in the art, including, but not limitedto sequencing, quantitative polymerase chain reaction (PCR), multiplexed(PCR), mass cytometry, fluorophore-inactivated multiplexedimmunofluorescence, hybridization-based methods, fluorescencehybridization-based methods, imaging, and any combinations thereof. Insome embodiments, the signals from the released identificationnucleotide sequences can be determined by electrophoresis-based methods.In some embodiments, the signals from the released identificationnucleotide sequences are not determined by electrophoresis-basedmethods.

In some embodiments, the signals from the released identificationnucleotide sequences can be detected by hybridization-based methods. Forexample, in some embodiments, the method can further comprise, prior tothe detecting in (c), coupling the released identification nucleotidesequences from (b) to a detection composition comprising a plurality ofreporter probes. Each reporter probe in the plurality can comprise (i) afirst target probe-specific region that is capable of binding a firstportion of the identification nucleotide sequence; and (ii) a detectablelabel that identifies the reporter probe. In these embodiments, signalsfrom the respective detectable labels of the reporter probes that arecoupled to the released identification nucleotide sequences can bedetected accordingly. Since the signals are distinguishable for eachrespective reporter probes that are bound to the identificationnucleotide sequences, target-binding molecules can be correspondinglyidentified, thereby detecting a plurality of target molecules in thesample.

In some embodiments where reporter probes are used in the methodsdescribed herein, the detectable label of the reporter probes cancomprise one or more labeling molecules that create a unique signal foreach reporter probe. For example, a unique signal can be an opticalsignal. The optical signal can be a light-emitting signal or a series orsequence of light-emitting signals. In some embodiments, labelingmolecules for generation of an optical signal can comprise one or aplurality of a fluorochrome moiety, a fluorescent moiety, a dye moiety,a chemiluminescent moiety, or any combinations thereof.

In some embodiments, the detection composition used in the methodsdescribed herein can additionally or alternatively comprise a pluralityof capture probes. Each capture probe can comprise (i) a second targetprobe-specific region that is capable of binding to a second portion ofthe identification nucleotide sequence; and (ii) an affinity tag. Theaffinity tag of the capture probe is generally used to permitimmobilization of the released identification nucleotide sequences, uponcoupling to the detection composition, onto a solid substrate surface.In some embodiments, immobilization of the released identificationnucleotide sequences can provide distinguishable spatial signals thatidentify the capture probes coupled to the released identificationnucleotide sequences. Examples of a solid substrate include, but are notlimited to, a microfluidic device, a cartridge, a microtiter plate, atube, and an array.

In some embodiments, the detection method in (d) does not requireamplification of the released identification nucleotide sequences, firsttarget probe-specific region, or the second target probe-specificregion. Amplification-free detection methods can minimize any bias orerrors introduced during amplification, e.g., due to varyingamplification efficiencies among the nucleotide sequences.

In some embodiments, identification nucleotide sequences of the targetprobes described herein can be selected or designed such that they donot cross-react with any nucleic acid sequence in a genome of a subject,whose sample is being evaluated. Thus, the identification nucleotidesequences used to detect target molecules from a subject's sample can beselected or designed based on nucleotide sequences of a species or genusthat is different from the subject. By way of example only, in someembodiments, the identification nucleotide sequences for use in ananimal's sample (e.g., a mammal such as a human) can be derived from aplant genome. In one embodiment, the identification nucleotide sequencesfor use in a human's sample can be derived from a potato genome. In someembodiments, the identification nucleotide sequences can have sequencesselected from Table 2 (SEQ ID NO: 1 to SEQ ID NO: 110), or a fragmentthereof.

Generally, identification nucleotide sequences of the target probes canhave any sequence length and can vary depending on a number of factors,including, but not limited to detection methods, and/or the number oftarget molecules to be detected. For example, in some embodiments, thelength of the identification nucleotide sequences can increase toprovide sufficient identification of a large number of target moleculesin a sample. In some embodiments where a hybridization-based method isused to detect identification nucleotide sequences, the identificationnucleotide sequences can have a length sufficient to provide reliablebinding to complementary reporter probes and to generate detectablesignals. In some embodiments, the identification nucleotide sequencescan have a length of about 30-100 nucleotides. In some embodiments, theidentification nucleotide sequences can have a length of about 70nucleotides.

The cleavable linker coupling a target-binding molecule to anidentification nucleotide sequence in a target probe can permit releaseof the identification nucleotide sequence from the target probe uponbinding to a target molecule such that the released identificationnucleotide sequence can then be detected. Cleavable linkers are known inthe art, of which examples include, but are not limited to the ones thatare sensitive to an enzyme, pH, temperature, light, shear stress,sonication, a chemical agent (e.g., dithiothreitol), or any combinationthereof. In some embodiments, the cleavable linker can be sensitive tolight and enzyme degradation.

In some embodiments, the cleavable linker does not comprise apolynucleotide sequence (e.g., a single-stranded polynucleotidesequence) that is complementary (for basepairing) to at least a portionof the identification nucleotide sequence. That is, in theseembodiments, the identification nucleotide sequence is not released fromthe complex by detaching from the complementary polynucleotide sequencecoupled to a target-binding molecule. Accordingly, in some embodiments,a target probe comprises (i) a target-binding molecule that specificallybinds to the target molecule present in the sample; (ii) anidentification nucleotide sequence that identifies the target-bindingmolecule; and (iii) a cleavable, non-hybridizable linker between thetarget-binding molecule and the identification nucleotide sequence.

In some embodiments, the cleavable, non-hybridizable linkers can beselected from the group consisting of hydrolyzable linkers, redoxcleavable linkers, phosphate-based cleavable linkers, acid cleavablelinkers, ester-based cleavable linkers, peptide-based cleavable linkers,photocleavable linkers, and any combinations thereof. In someembodiments, the cleavable linker can comprise a disulfide bond, atetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzylgroup, a nitoindoline group, a bromo hydroxycoumarin group, a bromohydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoingroup, or any combinations thereof.

In some embodiments, the cleavable, non-hybridizable linker can comprisea photocleavable linker. Any art-recognized photocleavable linker can beused for the target probes described herein. Exemplary photocleavablelinker is selected from the group consisting of molecules (i)-(xiv) andany combinations thereof, wherein the chemical structures of themolecules (i)-(xiv) are shown as follows:

where each of the black dots in each molecule represents a connecting orcoupling point that connects, directly or indirectly, to atarget-binding molecule described herein or an identification nucleotidesequence described herein. The connecting point can be a bond, orcomprise an atom, a molecule, and/or a linker described herein. In someembodiments, the connecting point is a bond.

In some embodiments, the photocleavable linker can comprise the molecule(xiv).

In some embodiments where a photocleavable linker is used, theidentification nucleotide sequences can be released from the boundtarget probes by exposing the bound target probes to a light of aspecified wavelength. In some embodiments, ultraviolet light can be usedto release identification nucleotide sequences from bound target probes.

In some embodiments, the method can further comprise, prior tocontacting the sample with target probes, separating target cells frominterfering cells in the sample. Methods to separate target cells frominterfering cells are known in the sample, e.g., based on cell surfaceproteins that distinguish target cells from interfering cells. By way ofexample only, target cells or interfering cells can be labeled withligands that target specific cells of interests (e.g., cell-specificantibodies). In some embodiments where the cell-specific ligands arefluorescently labeled, the labeled cells can then be sorted, e.g., byflow cytometry. Alternatively, if the cell-specific ligands are attachedto magnetic particles, the labeled cells with bound magnetic particlescan be isolated from the sample by magnetic separation.

Target cells can be prokaryotic or eukaryotic, including microbes (e.g.,bacteria, fungus, virus, and/or pathogens. In some embodiments, thetarget cells can comprise normal cells, diseased cells, mutant cells,germ cells, somatic cells, and/or rare cells. Example of rare cellsinclude, without limitations, circulating tumor cells, fetal cells, stemcells, immune cells, clonal cells, and any combination thereof. In someembodiments, the target cells can comprise tumor cells. In someembodiments, the tumor cells can be derived from a tissue biopsy, a fineaspirate or a liquid biopsy (e.g., peritoneal, pleural, cerebrospinalfluid, and/or blood), a mucosal swap, a skin biopsy, a stool sample, orany combinations thereof. In some embodiments, whole cells and/or celllysates can be used in the methods and/or systems described herein todetect a plurality of target molecules in a sample. In some embodiments,the whole cells can be obtained from a fixed cell or tissue sample.

Typically, signals detected from the identification nucleotide sequencesof the target probes corresponding to target molecules can be comparedto a control reference to account for any non-specific binding.Accordingly, in some embodiments, the composition added to the samplecan further comprise a plurality of control probes. Each control probein the plurality can comprise: (i) a control-binding molecule thatspecifically binds to one control molecule in the sample; (ii) anidentification control sequence that identifies the control-bindingmolecule; and (iii) a cleavable linker between the control-bindingmolecule and the identification control sequence. The control-bindingmolecule can bind to a control protein present in a sample. Non-limitingexamples of control proteins include housekeeping proteins, control IgGisotypes, mutant non-functional or non-binding proteins, and anycombinations thereof.

Signals from the control probes can then be used to threshold thesignals from the target probes. Accordingly, in some embodiments, themethod can further comprise thresholding the target signals. In someembodiments, the target signals can be thresholded on the basis ofnonspecific binding. In one embodiment, the threshold is generally setto be greater than that of the signals from the non-specific binding. Insome embodiments, the threshold can be determined by using standarddeviation and measurement error from at least one or more controlproteins.

In some embodiments, the method can further comprise quantifying thesignals (e.g., signals that are above the pre-determined threshold) bynormalizing the signals associated with the target probes by the signalsassociated with the control probes. In one embodiment, the signals isquantified and expressed as number of identification nucleotidesequences detected per target-binding agent

In some embodiments, the method can further comprising extracting anucleic acid molecule for the same sample for a nucleic acid analysis.Examples of a nucleic acid detection and analysis can include, but arenot limited to sequencing, quantitative polymerase chain reaction (PCR),multiplexed PCR, DNA sequencing, RNA sequencing, de novo sequencing,next-generation sequencing such as massively parallel signaturesequencing (MPSS), polony sequencing, pyrosequencing, Illumina (Solexa)sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoballsequencing, Heliscope single molecule sequencing, single molecule realtime (SMRT) sequencing, nanopore DNA sequencing, sequencing byhybridization, sequencing with mass spectrometry, microfluidic Sangersequencing, microscopy-based sequencing techniques, RNA polymerase(RNAP) sequencing, or any combinations thereof.

While the methods described herein are described in the context wherethe identification nucleotide sequences are released from bound targetprobes before detection, in some embodiments, the identificationnucleotide sequences do not need to be released from the bound targetprobes. Accordingly, in some embodiments, the methods described hereincan also apply when the identification nucleotide sequences remain boundto target probes during detection.

Various embodiments of the methods described herein can be carried outin one or more functional modules in a system or a computer system asdescribed herein. Accordingly, another provided herein relates to asystem for multiplexed detection of a plurality of target molecules in asample. For example, the system comprises:

(a) at least one sample processing module comprising instructions forreceiving said at least one test sample comprising a sample and aplurality of target probes, wherein each target probe in the pluralitycomprises:

-   -   i. a target-binding molecule that specifically binds to one        target molecule in the sample;    -   ii. an identification nucleotide sequence that identifies the        target-binding molecule; and    -   iii. a cleavable linker between the target-binding molecule and        the identification nucleotide sequence; and        wherein the at least one sample processing module further        comprises instructions for releasing the identification        nucleotide sequences from the target probes that are bound to        target molecules in the sample;

(b) a signal detection module comprising instructions for detectingsignals from the released identification nucleotide sequences;

(c) at least one data storage module comprising instructions for storingthe detected signals from (b) and information associated withidentification nucleotide sequences of the target probes;

(d) at least one analysis module comprising instructions for determiningthe presence of one or more target molecules in the sample based on thedetected signals; and

(e) at least one display module for displaying a content based in parton the analysis output from said analysis module, wherein the contentcomprises a signal indicative of the following: (i) the presence of oneor more target molecules in the sample, (ii) the absence of one or moretarget molecules in the sample, and/or (iii) expression levels of one ormore target molecules in the sample.

In some embodiments, the analysis module can further compriseinstructions for (i) identifying the detectable probes of the reporterprobes that correspond to the detected signals; (ii) identifying theidentification nucleotide sequences of the target probes that correspondto the detectable probes based on the first target probe-specificregions of the reporter probes; and (iii) identifying the target-bindingmolecules that correspond to the identification nucleotide sequences,thereby determining the presence of one or more target molecules in thesample.

In some embodiments, the content can be displayed on a computer display,a screen, a monitor, an email, a text message, a website, a physicalprintout (e.g., paper), or provided as stored information in a datastorage device.

Kits, e.g., for multiplexed detection of a plurality of different targetmolecules from a sample, are also provided herein. In some embodiments,the kit comprises:

(a) a plurality of target probes, wherein each target probe in theplurality comprises:

-   -   i. a target-binding molecule that specifically binds to one        target molecule in the sample;    -   ii. an identification nucleotide sequence that identifies the        target-binding molecule; and    -   iii. a cleavable linker between the target-binding molecule and        the identification nucleotide sequence; and

(b) a plurality of reporter probes, wherein each reporter probecomprises:

-   -   i. a first target probe-specific region that is capable of        binding a first portion of the identification nucleotide        sequence; and    -   ii. a detectable label that identifies the reporter probe.

In some embodiments, the detectable label of the reporter probes cancomprise one or more labeling molecules that create a unique signal foreach reporter probe. An exemplary unique signal can be an opticalsignal. The optical signal can comprise one or a series or a sequence oflight-emitting signals. In these embodiments, non-limiting examples ofthe labeling molecules include fluorochrome moieties, fluorescentmoieties, dye moieties, chemiluminescent moieties, and any combinationsthereof.

In some embodiments, the kit can further comprise a plurality of captureprobes, wherein each capture probe comprises (i) a second targetprobe-specific region that is capable of binding a second portion of theidentification nucleotide sequence; and (ii) an affinity tag.

In some embodiments, the kit can further comprise a plurality of controlprobes, wherein each control probe in the plurality comprises:

-   -   (i) a control-binding molecule that specifically binds to one        control molecule in the sample;    -   (ii) an identification control sequence that identifies the        control-binding molecule; and    -   (iii) a cleavable linker between the control-binding molecule        and the identification control sequence.

In some embodiments, the kit can further comprise at least one reagentfor use in one or more embodiments of the methods or systems describedherein. Reagents that can be provided in the kit can include at leastone or more of the following: a hybridization reagent, a purificationreagent, an immobilization reagent, an imaging agent, a cellpermeabilization agent, a blocking agent, a cleaving agent for thecleavable linker, primers for nucleic acid detection, nucleic acidpolymerase, and any combinations thereof.

In some embodiments, the kit can further include a device for use in oneor more embodiments of the methods and/or systems described herein. Insome embodiments, the device can comprise a surface for immobilizationof the capture probes upon coupling to the identification nucleotidesequences. In some embodiments, the device can comprise a microfluidicdevice for separating target cells from interfering cells as describedherein.

The methods, systems and kits described herein can be used to detect anytarget molecules present in a sample provided that appropriatetarget-binding agents are used in the target probes employed in themethods described herein. Exemplary target molecules which can bedetected by the methods, systems and kits described herein include, butare not limited to proteins, peptides, metabolites, lipids,carbohydrates, toxins, growth factors, hormones, cytokines, cells (e.g.,eukaryotic cells, prokaryotic cells, and microbes), and any combinationsthereof. In some embodiments, the target molecules to be detected can beextracellular or secreted molecules. In some embodiments, the targetmolecules to be detected can be intracellular, e.g., cytoplasmicmolecules or nuclear molecules.

To detect intracellular molecules (e.g., intracellular proteins), thetarget cells in the sample can be permeabilized or lysed such thattarget probes can contact the target intracellular molecules for furtherprocessing and analysis.

In some embodiments, the methods, systems and kits described herein canenable measurements of at least two target molecules of different types.For example, the methods, systems, and kits described herein can be usedto measure, for example, nucleic acid molecules and proteins, orproteins and metabolites, or proteins and lipids. The measurements of atleast two target molecules of different types can be performedsimultaneously or sequentially. In another embodiment, by releasingidentification nucleotide sequences from bound target molecules (e.g.,proteins), genetic material and the identification nucleotide sequencescan be concurrently extracted from a single sample, enabling analyses ofprotein-DNA-RNA interrelationships.

By way of example only, the methods, systems and kits described hereinapplied to a sample can preserve genetic materials in a sample whiledetecting other non-genetic target materials in the same sample.Accordingly, in some embodiments, the methods, systems and/or kitsdescribed herein for detection of non-genetic target molecules (e.g.,but not limited to proteins) can be used in combination with a nucleiacid analysis for genetic materials, for example, to study thenon-genetic target molecules (e.g., but not limited to proteins) thatinteract with genetic materials or genetic regulatory elements. In theseembodiments, the methods and systems described herein for detecting aplurality of target molecules in a sample as described herein canfurther comprise extracting a nucleic acid molecule from the same samplein which target molecules are to be detected. In some embodiments, themethods and systems described herein can further comprise subjecting theextracted nucleic acid molecule to a nucleic acid analysis. Variousmethods can be used for nucleic acid analysis, including, but notlimited to sequencing, next generation sequencing, quantitativepolymerase chain reaction (PCR), multiplexed PCR, DNA sequencing, RNAsequencing, de novo sequencing, next-generation sequencing such asmassively parallel signature sequencing (MPSS), polony sequencing,pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ionsemiconductor sequencing, DNA nanoball sequencing, Heliscope singlemolecule sequencing, single molecule real time (SMRT) sequencing,nanopore DNA sequencing, sequencing by hybridization, sequencing withmass spectrometry, microfluidic Sanger sequencing, microscopy-basedsequencing techniques, RNA polymerase (RNAP) sequencing, fluorescencehybridization-based technology (e.g., but not limited to nanoStringnCounter® hybridization technology), any art-recognized nucleic aciddetection methods, or any combinations thereof.

In some embodiments, after a sample and/or non-genetic target moleculeshave been labeled with a plurality of target probes described herein,the identification nucleotide sequences of the target probes can bereleased from the bound non-genetic target molecules simultaneously withextraction of nucleic acid molecules (cells' genetic materials) from thesame labeled sample. In these embodiments, both the nucleic acidmolecules of interest and the identification nucleotide sequences can bedetected simultaneously in a single sample mixture. In one embodiment,both the nucleic acid molecules of interest and the identificationnucleotide sequences can be detected simultaneously in a single samplemixture using nanoString nCounter® analysis system, for example, asdescribed in U.S. Pat. No. 8,415,102, the content of which isincorporated herein by reference. Other art-recognized methods fornucleic acid analyses as described herein can also be used forsimultaneous detection of both nucleic acid molecules of interest(cells' genetic materials) and released identification nucleotidesequences from bound non-genetic target molecules.

In alternative embodiments, nucleic acid molecules can be extracted froma first portion of a sample, while non-genetic target molecules can beindependently derived or obtained from a second portion of the samesample. In these embodiments, the nucleic acid molecules of interest andthe non-genetic target molecules can be detected separately to determineexpression levels of the nucleic acid molecules (cells' geneticmaterials) of interest and non-genetic target molecules (e.g., but notlimited to proteins) in the same sample. The nucleic acid molecules ofinterests can be subjected to any art-recognized nucleic acid analysis,while the non-genetic target molecules can be detected through detectingand identifying the corresponding identification nucleotide sequencesreleased from the target probes using the methods, systems and/or kitsdescribed herein.

In some embodiments, the methods, systems and kits described herein canbe adapted to measure proteins and nucleic acid molecules in the samesample. For example, the proteins can be detected by one or moreembodiments of the target probes described herein, while the nucleicacid molecules can be detected by any methods known in the art, therebycreating an integrated expression profiling for the sample, which canprovide information on interaction between the proteins and the nucleicacid molecules, e.g., genetic regulatory elements such as microRNAs.

The methods, systems and kits described herein can be used in anyapplications where detection of a plurality of target molecules in asample is desirable. For example, a sample can be a biological sample,or a sample from an environmental source (e.g., water, soil, foodproducts, and/or ponds).

The inventors have demonstrated that, in one embodiment, an antibodybarcoding with photocleavable DNA (ABCD) platform described herein canenable analysis of hundreds of proteins from a single cell or a limitednumber of cells, e.g., from minimally invasive fine-needle aspirates(FNAs). Accordingly, samples amenable to the methods described hereincan comprise less than 500 cells or fewer. In some embodiments, thesample can be a single-cell sample. In some embodiments, the sample cancomprise cells isolated from a fine-needle aspirate.

Where the sample is a biological sample, in some embodiments, themethods, systems and kits described herein can be used in personalizedtreatment. For example, a biological sample can be collected from anindividual subject who is in need of a treatment for a condition. Usingthe methods, systems and/or kits described herein, an expression profileof target molecules associated with the subject's condition can begenerated to identify one or more therapeutic targets for the subject,thereby identifying a treatment regimen for the subject.

In some embodiments, the methods, systems and kits described herein canbe used in monitoring response of a subject to a treatment for his/hercondition. For example, biological sample(s) can be collected from thesubject prior to and/or over the course of the treatment. Using themethods, systems and/or kits described herein, expression profiles oftarget molecules associated with the subject's condition before and/orover the course of the treatment can be generated for comparison todetermine any changes in expression levels of the target molecules inthe subject, thereby monitoring the treatment response in the subject.

In some embodiments, the methods, systems and kits described herein canbe used in diagnosing a condition in a subject. For example, abiological sample can be collected from a subject who is at risk for acondition. Using the methods, systems and/or kits described herein, anexpression profile of target molecules associated with the condition tobe diagnosed can be generated for comparison with one or more referenceexpression profiles (e.g., corresponding to a normal healthy subjectand/or a subject having the condition to be diagnosed), therebydetermining whether the subject is at risk for the condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows an exemplary scheme of a multiplexed protein analysisin single cells in accordance with one or more embodiments of themethods described herein. (FIG. 1A) Cells were harvested from cancerpatients by FNAs. In this case, a heterogeneous population ofEpCAM-positive cancer cells (green) is displayed alongside mesothelialcells (red) with nuclei shown in blue (Hoechst) from an abdominal cancerFNAs. Cancer cells were enriched and isolated via magnetic separation inpolydimethylsiloxane (PDMS) microfluidic devices with herringbonechannels using both positive (for example, EpCAM+/CK+) and negative (forexample, CD45−) selection modes. (FIG. 1B) Cells of interest wereincubated with a cocktail of DNA-conjugated antibodies containing aphotocleavable linker (FIG. 2A) to allow DNA release after exposure toultraviolet light. (FIG. 1C) DNA-antibody conjugates released from lysedcells (FIG. 3) were isolated using size separation and IgG pull-down.Released “alien” DNA barcodes were processed with a fluorescent DNAbarcoding platform (NanoString). Fluorescent barcodes were hybridizedand imaged using a CCD camera. The quantified barcodes were translatedto protein expression levels by normalizing to DNA per antibody andhousekeeping proteins and subtracting nonspecific binding from controlIgGs. A representative profile of SKOV3 ovarian cancer cell lines showshigh CD44 and high Her2 expression, characteristic of this cell line.

FIGS. 2A-2B shows an exemplary scheme of DNA-antibody conjugation. (FIG.2A) Various linker strategies were investigated to conjugate DNA toantibodies. In some embodiments, the photocleavable linker (PCL) wasselected owing to its better cleavage efficiency compared with DTT,tetrazine-trans-cyclooctene (via click chemistry, linker 1), and Traut'sreagent (linker 2). Linker cleavage was tested by measuring released DNAvia the NanoString platform. Data are averages of two independenttrials. **P<0.01, paired t-test. (FIG. 2B) Linking DNA to an antibodyvia the PCL. The linker was first reacted with the amine (—NH₂) groupson the antibody. After excess small molecule was removed, thiolated DNAwas added at 10-fold excess to the antibody-linker mix. The finalantibody-DNA chimera was purified via both size separation andIgG-specific pulldown. DNA could subsequently be released from theantibody by photocleavage at a specific wavelength (365 nm).

FIG. 3 shows experimental data directed to optimization of lysis andblocking methods. (Methods A to D) Four different lysis and blockingmethods were used to recover DNA from labeled cells. Lysate conditionsincluded: (Method A) Proteinase K+PKD lysis buffer; (Method B)Proteinase K+ATL lysis buffer; (Method C) ATL lysis buffer alone; and(Method D) UV cleavage alone (no cell lysis). The lysate conditions weretested in duplicate (x-axis) measuring DNA signal (y-axis) and differentintracellular proteins (z-axis). The best reaction condition was methodB (Proteinase K+ATL lysis buffer), with a 20% increase in signal overmethods (Methods A and C).

FIG. 4 is a graph showing the readouts of DNA per antibody for eachtarget molecule. The number of alien DNA fragments per antibody wasmeasured by Nanostring method (shown in graph) and independentlyconfirmed by ssDNA quantification and Qubit protein measurement. Dataare displayed from triplicate measurements±SEM.

FIG. 5 shows a multiplexed protein profiling of a human breast cancercell line. Representative example of 88 different antibodies spanningcancer-relevant pathways (color-coded) profiled in triplicate (mean±SEM)on the MDAMB-231 triple-negative breast cancer cell line. DNA countswere converted to protein binding by normalizing to the amount of DNAper antibody. Nonspecific binding from expression of six control IgGswas subtracted, and expression was normalized by housekeeping proteinsCox IV, histone H3, tubulin, actin, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (far right). AU, arbitrary units; EMT,epithelial-to-mesenchymal transition.

FIGS. 6A-6B are graphs showing effect of permeabilization schemes onantibody labeling. (FIG. 6A) Methanol and saponin permeabilization weresimilar for both intracellular and nuclear proteins. Representativeexamples are graphed, such as phospho-histone H3 (pH3), epithelial celladhesion molecule (EpCAM), phosphorylated Src (pSRC), and phosphorylatedglycogen synthase kinase 3β(pGSK3b). (FIG. 6B) Nonspecific binding wasmuch higher with methanol permeabilization.

FIGS. 7A-7B are experimental data showing comparison of unmodifiedantibodies to DNA-antibody conjugates. (FIG. 7A) Antibody-DNA conjugatesshow good correlation against unconjugated, native antibodies, asdetermined by flow cytometry. Experiments were performed on multiplecell lines. A representative example is shown with a head-to-headcomparison of multiple antibodies on the human SKOV3 cell line(R²=0.92). (FIG. 7B) Protein expression detected in different celllysates showed similar patterns of expression whether detected byunmodified antibodies or DNA conjugates. This held true for p53 andphospho-S6RP (immunoblotting), and Ki67 (dot blot).

FIGS. 8A-8C show validation data of DNA-antibody conjugates. (FIG. 8A)Concordance between two different antibody clones of EpCAM (MOC-31 and158206) when conjugated to separate DNA barcodes. The antibodies wereassayed across multiple patient samples (n=22). (FIG. 8B) Antibodyexpression was measured when cell lines were stained with a singleantibody as compared to a cocktail (80+ antibodies). Data were collectedfrom 5 antibodies (CD44, EGFR, 53BP1, p-S6RP, rabbit IgG) on 3 differenthuman cell lines (MDA-MB-231, MDA-MB-436, and A431). The experiment wasrepeated in duplicate and each data point corresponds to one marker on agiven cell line. Expression measurements were calculated by normalizedDNA counts for the same number of cells. (FIG. 8C) Changes in markerexpression before and after treatment were assayed and quantified usingboth the method in the Examples (ABCD platform) and an independentimmunofluorescence screen (standard error is shown from biologicaltriplicate).

FIG. 9 is a set of graphs showing that protein marker expressioncorrelates with flow cytometry. Multiple markers (CD44, Her2, EGFR,CA19-9, Keratin 7, and Muc 1) were screened across multiple cell lines(SK-OV-3, ES-2, OVCA429, UCI-107, UCI-101, TOV-112D, TOV-21G, andA2780). Each data point represents expression derived from NanoStringDNA counts or flow cytometry for a particular cell line. Expressionvalues were normalized by housekeeping proteins GAPDH, tubulin, andactin. Cell lines with measurements below that of the negative control(IgG antibodies) either on flow cytometry or Nanostring were excluded.These measurements were compared to independently performed flowcytometry measurements, which were calculated from the mean fluorescenceintensity (signal/background), where the background was the secondaryantibody without the primary antibody. The inset shows the log-log plotof the data.

FIGS. 10A-10C are experimental data on detection sensitivity using ahuman epidermal cancer cell line. (FIG. 10A) A bulk sample of 500,000cells from the epidermoid carcinoma cell line A431 was lysed andprocessed as shown in FIGS. 1A-1C. Dilutions corresponding to 5, 15, and50 cells were then compared to the bulk measurement. (FIG. 10B)Correlation values for single A431 cells selected by micromanipulationare compared to the bulk measurements (500,000 cells). (FIG. 10C)Protein expression profiles (log₂ expression values) of four singlecells compared with the bulk sample. Correlations were highlysignificant when comparing all single cells to bulk measurements(P<0.0001, paired t test; GraphPad Prism 6.0).

FIGS. 11A-11B are experimental data showing single-cell variability intreated and untreated A431 cells. (FIG. 11A) Single cell measurements inhuman A431 cell lines that were either treated or untreated with theEGFR inhibitor gefitinib were clustered based on a correlation metric(MatLab). (FIG. 11B) Pairwise t-tests for four markers (FDR=0.1,***P<0.001, GraphPad Prism), are shown. Markers that were mostsignificant are shown, as well as phosphor-EGFR, which is the primarytarget of gefitinib inhibition. The distribution between signals fromuntreated cells (blue) and treated cells (yellow) are shown. Each pointrepresents expression levels calculated from a single cell, and the meanand standard deviation are shown in the box plot.

FIGS. 12A-12B show experimental data based on a single-cell proteinanalysis in a patient sample. An FNA was obtained from a patient withbiopsy-proven lung adenocarcinoma. (FIG. 12A) Eleven harvested cellswere analyzed individually, and protein expression levels in each cell(y axis) were correlated with expression levels from the bulk tumorsample (x axis). Each data point represents the expression for a givenmarker (n=85 markers, 3 below detection threshold). (FIG. 12B) SpearmanR correlation coefficient values for each of the single cells in (FIG.12A) relative to each other and to the bulk measurement.

FIG. 13 shows interpatient heterogeneity in lung cancer. FNAs wereobtained from six patients with biopsy-proven lung adenocarcinoma, andbulk samples (˜100 cells each) were processed as shown in FIGS. 1A-1Cwith 88 barcoded antibodies. Expression data were log 2-normalized byrow to show differences between each patient. Expression profiles wereheterogeneous despite the identical histological type: Upon geneticanalysis, patients 1 and 2 had EGFR exon 19 amplification and T790Mmutations, patient 3 had an exon 20 EGFR mutation, patient 4 had an EGFRL858R mutation and an additional BRAF mutation, patient 5 had a KRASmutation, and patient 6 had an EML4-ALK translocation.

FIGS. 14A-14B show experimental data on effect of different therapies onprotein expression profiles in MDA-MB-436 triple-negative breast cancercell line. (FIG. 14A) MDA-MB-436 cells were treated with differentagents, and marker proteins were measured. Unsupervised hierarchicalclustering based on Euclidean distance grouped drug treatments by theirmechanisms of action (molecularly targeted versus DNA damaging) andprimary targets [EGFR for gefitinib/cetuximab and mammalian target ofrapamycin (mTOR)/PI3K for PKI-587]. Data show the log₂ fold change ofmarker expression in treated compared to untreated cells for n=84markers. All experiments were performed in triplicate. (FIG. 14B)Correlating drug sensitivity of four different cell lines with proteomicprofile changes after treatment with cisplatin and olaparib. IC50 values(black bars) were calculated on the basis of viability curves (FIG.15A). The cell profile change after treatment is represented by thenumber of significant markers (gray bars) that were identified by apairwise t test of treated versus untreated samples (FDR=0.1).

FIGS. 15A-15E are graphs showing protein marker changes correlate withdrug sensitivity. Human ovarian carcinoma (A2780, OVCAR429) and breastcancer (MDA-MB-436, MDA-MB-231) cell lines react differently tochemotherapy. Those with increased sensitivity to a drug are expected toshow a greater degree of change in their cell profiles. (FIG. 15A)Sensitivity was determined by IC50 values calculated from MTS viabilitycurves in biological triplicate for each cell line as shown. Exactvalues and the fit of the viability curves were determined by GraphPadPrism 5.0 (dose-response curve). (FIG. 15B) Data of a control studywhere cell lines were treated with cetuximab, which resulted in druginhibition. (FIGS. 15C-15E) Changes across a selected panel of severalDNA damage markers (pH2A.X, Ku80, pChk2, pChhk1), apoptosis markers(cleaved PARP, cleaved caspase 7), and other mechanisms commonlyassociated with platinum treatment (pERK, Bim). Data are means±SEM,performed in triplicate.

FIGS. 16A-16D show that taxol treatment and dose response screens inhuman HT1080 cells in vitro. (FIG. 16A) Select marker changes from doseresponse taxol treatment are displayed with DNA barcoding profiles withstandard error from biological duplicate. (FIG. 16B) EC50 values fromthe dose response curves are displayed along with R² values. (FIG. 16C)Markers that significantly differed from untreated (pairwise multiplet-test, FDR=0.2) were shown to have a dose-dependent response to taxoltreatment. (FIG. 16D) The markers that significantly different betweenuntreated and treated conditions are shown in a venn diagram. CDCP1 wassignificantly different at all doses.

FIGS. 17A-17B show expression profilings of various cancer patients formonitoring and predicting treatment response in patients receiving PI3Kinhibitors. (FIG. 17A) Profiles of five drug-naïve cancer patients areshown with clustering based on correlation metrics with weightedlinkage. The dotted box shows the cluster including the marker that bestseparated responders and nonresponders (H3K79me2). Other markers in thecluster include pS6RP (a downstream target of PI3K), pH2A.X (DNA damagemarker), PARP (DNA repair protein), and 4EBP1 (protein translation).(FIG. 17B) Four patients with biopsy-proven adenocarcinoma were treatedwith PI3Ki, and primary cancers were biopsied before and aftertreatment. The heat map is a pre-post treatment difference map showinglog 2 fold changes in protein expression (normalized by row to highlightdifferences between patients). Patient segregation is by correlationdistance metric (weighted linkage). The patient in the third columnreceived a higher dose of the PI3Ki (400 mg, twice daily) than thepatient in the fourth column (150 mg, twice daily).

FIGS. 18A-18B are block diagrams showing exemplary systems for use inthe methods described herein, e.g., for multiplexed detection of targetmolecules in a sample.

DETAILED DESCRIPTION

Immunohistochemistry-based clinical diagnoses generally require invasivecore biopsies and use a limited number of protein stains to identify andclassify cancers. Fine-needle aspirates (FNAs) employ thin needles toobtain cells from tumor masses and the procedure is thus minimallyinvasive. While FNAs can give tremendous insight into malignancy, thenumber of cells in the FNAs is so small (compared to core biopsy) thatcurrent technologies for protein analysis, such as immunohistochemistry,are insufficient. Embodiments of various aspects described herein are,in part, based on the development of a scalable method that not onlyallows analysis of a plurality of proteins from a limited amount ofsample, e.g., FNAs, but also preserves genetic material from the samesample to enable simultaneous measurements of proteins and geneticmaterials (e.g., DNA, RNA, epigenetic and microRNAs). In one embodiment,the method relies on DNA-barcoded antibody sensing, wherebarcodes-single strands of DNA- can be photocleaved and detected usingfluorescent complementary probes without any amplification steps, and isreferred to as an antibody barcoding with photocleavable DNA (ABCD)platform herein. Unlike the protein detection method described in U.S.Pat. App. Pub. No. US 2011/0086774, the DNA barcode and the antibodythat the inventors developed is coupled together through a cleavable,non-hybridizable linker, not a hybridizable linker that is reversiblyhybridized (e.g., by basepairing) to a portion of the DNA barcode. Inaddition, detection of a target protein is based on binding of a singleDNA-barcoded antibody to the target protein, which is different from theprotein detection method described in U.S. Pat. App. Pub. No. US2011/0086774, where two antibodies (one for immobilization to a solidsubstrate, e.g., a bead, and another for detection purpose) are requiredfor binding to different regions of the target protein.

To demonstrate the capability of the ABCD platform, inventors isolatedcancer cells within the FNAs of patients and exposed these cells to amixture of about 90 DNA-barcoded antibodies, covering the hallmarkprocesses in cancer (for example, apoptosis and DNA damage). Theinventors discovered that the single-cell protein analysis of thepatients' FNAs showed high intratumor heterogeneity, indicating theability of the ABCD platform to perform protein profiling on rare singlecells, including, but not limited to circulating tumor cells. Further,the inventors discovered that patients who showed identicalhistopathology yet showed patient heterogeneity in proteomic profiling,indicating the ability of the ABCD platform to identify personalizedtargets for treatment. By profiling and clustering protein expression inpatients' samples, the inventors also showed use of the ABCD platform tomonitor and predict treatment response in patients receivingchemotherapy, e.g., kinase inhibitors. The ABCD platform for generatinga protein profiling is scalable and can be extended to detect othertarget molecules, e.g., metabolites and lipids. Not only can the ABCDplatform measure protein quantitatively, but the ABCD platform can alsoenable simultaneous measurements of at least 90 different proteins ormore (e.g., about 100-200 different proteins) in a single sample.Further, because of the high sensitivity of the ABCD platform, the ABCDplatform can enable detection of rare proteins, e.g., proteins that arenot generally highly-expressed, or not easily accessible or extracted,such as intracellular proteins. Accordingly, various aspects describedherein provide for methods, systems and kits for detecting and/orquantifying a plurality of target molecules from a sample, as well astheir uses thereof in various applications, e.g., diagnosis, prognosis,personalized treatment, and/or treatment monitoring.

Methods for Detecting or Quantifying a Plurality of Target Molecules ina Sample

In one aspect, provided herein is a method for detecting a plurality oftarget molecules in a sample. The method comprises (a) contacting asample with a composition comprising a plurality of target probes,wherein each target probe in the plurality comprises: (i) atarget-binding molecule that specifically binds to a target molecule ora distinct target molecule in the sample; (ii) an identificationnucleotide sequence that identifies the target-binding molecule; and(iii) a cleavable linker between the target-binding molecule and theidentification nucleotide sequence; (b) releasing the identificationnucleotide sequences from the bound target probes; and (c) detectingsignals from the released identification nucleotide sequences, whereinthe signals are distinguishable for the identification nucleotidesequences, thereby identifying the corresponding target-bindingmolecules and detecting a plurality of target molecules in the sample.

In some embodiments where each target probe in the plurality binds to adistinct target molecule, no two target probes in the plurality binds todifferent regions of the same target molecule.

Stated another way, the method comprises: (a) forming a plurality ofcomplexes in a sample, each complex comprising a target molecule and atarget probe bound thereto, wherein the target probe comprises (i) atarget-binding molecule that specifically binds to the target moleculepresent in the sample; (ii) an identification nucleotide sequence thatidentifies the target-binding molecule; and (iii) a cleavable linkerbetween the target-binding molecule and the identification nucleotidesequence; (b) releasing the identification nucleotide sequences from thecomplex; and (c) detecting signals from the released identificationnucleotide sequences, wherein the signals are distinguishable for theidentification nucleotide sequences, thereby identifying thecorresponding target-binding molecules and detecting a plurality oftarget molecules in the sample. In some embodiments, the cleavablelinker is not pre-hybridized (e.g., by basepairing) to any portion ofthe identified nucleotide sequences.

In some embodiments, e.g., cell assay, each complex comprising a targetmolecule and a target probe bound thereto does not require two or moretarget probes of different kinds bound to the same target molecule,where each of the target probes binds to a different region of the sametarget molecule. For example, unlike the protein detection methoddescribed in the U.S. Pat. App. No. US 2011/0086774, each complexdescribed herein does not require both a first target probe binding to afirst region of a target molecule, and a second target probe binding toa second region of the same target molecule. Stated another way, in someembodiments, a single target probe as described herein binding to atarget molecule is sufficient for enablement of the methods describedherein. In these embodiments, the method described herein does notrequire another target probe binding to the same target molecule forattachment to a solid substrate (e.g., a bead), e.g., as described inthe U.S. Pat. App. No. US 2011/0086774.

In various embodiments of different aspects described herein, thecleavable linker does not comprise a polynucleotide sequence (e.g., asingle-stranded polynucleotide sequence) that is complementary (forbasepairing) to at least a portion of the identification nucleotidesequence. That is, in these embodiments, the identification nucleotidesequence is not released from the complex by detaching from thecomplementary polynucleotide sequence coupled to a target-bindingmolecule. Accordingly, in some embodiments, a target probe comprises (i)a target-binding molecule that specifically binds to the target moleculepresent in the sample; (ii) an identification nucleotide sequence thatidentifies the target-binding molecule; and (iii) a cleavable,non-hybridizable linker between the target-binding molecule and theidentification nucleotide sequence.

“Target probes” is described in detail in the following “Target Probes”section.

In some embodiments, the method can further comprise separating unboundtarget probes from target probes that are bound to the target moleculesin the sample.

As used herein, the term “bound target probes” refers to target probesbinding to target molecules in a sample.

In some embodiments, the method can further comprise, prior tocontacting the sample with target probes, separating target cells frominterfering cells in the sample. Methods to separate target cells frominterfering cells are known in the sample, e.g., based on cell surfaceproteins that distinguish target cells from interfering cells. By way ofexample only, target cells or interfering cells can be labeled withligands that target specific cells of interests (e.g., cell-specificantibodies). In some embodiments where the cell-specific ligands arefluorescently labeled, the labeled cells can then be sorted, e.g., byflow cytometry. Alternatively, if the cell-specific ligands are attachedto magnetic particles, the labeled cells with bound magnetic particlescan be isolated from the sample by magnetic separation. In someembodiments, the cell sorting or selection can be performed in amicrofluidic device. In some embodiments, methods for isolating targetcells or interfering cells from a sample as described in theInternational Pat. App. No. WO 2013/078332, the content of which areincorporated herein by reference, can be used in combination with themethods described herein.

Target cells can be prokaryotic or eukaryotic (including microbes suchas bacteria, fungi, virus and/or pathogens). In some embodiments, thetarget cells can comprise normal cells, diseased cells, mutant cells,germ cells, somatic cells, and/or rare cells. Example of rare cellsinclude, without limitations, circulating tumor cells, fetal cells, stemcells, immune cells, clonal cells, and any combination thereof. In someembodiments, the target cells can comprise tumor cells. In someembodiments, the tumor cells can be derived from a tissue biopsy, a fineaspirate or a liquid biopsy (e.g., peritoneal, pleural, cerebrospinalfluid, and/or blood), a mucosal swap, a skin biopsy, a stool sample, orany combinations thereof. In some embodiments, whole cells and/or celllysates can be analyzed by the methods described herein to detect aplurality of target molecules in a sample. In some embodiments, thewhole cells can be obtained from a fixed cell or tissue sample.

Exemplary target molecules which can be detected by the methodsdescribed herein include, but are not limited to proteins, peptides,metabolites, lipids, carbohydrates, toxins, growth factors, hormones,cytokines, cells, and any combinations thereof. In some embodiments, thetarget molecules to be detected can be extracellular or secretedmolecules. In some embodiments, the target molecules to be detected canbe intracellular, e.g., cytoplasmic molecules or nuclear molecules.

To detect intracellular molecules (e.g., intracellular proteins), thetarget cells in the sample can be permeabilized or lysed (e.g., with alysis buffer or a surfactant) such that target probes can contact thetarget intracellular molecules for further processing and analysis. Insome embodiments, the lysis buffer can comprise a protease. An exemplaryprotease is a protease K.

The identification nucleotide sequences can be released from the boundtarget probes using any methods known in the art, depending on the typesof the cleavable linkers. In some embodiments, the cleavable linker doesnot comprise a polynucleotide sequence (e.g., a single-strandedpolynucleotide sequence) that is complementary (for basepairing) to atleast a portion of the identification nucleotide sequence. That is, inthese embodiments, the identification nucleotide sequence is notreleased from the complex by detaching from the complementarypolynucleotide sequence (hybridizable linker) coupled to atarget-binding molecule. Cleavable, non-hybridizable linkers are knownin the art, of which examples include, but are not limited to the onesthat are sensitive to an enzyme, pH, temperature, light, shear stress,sonication, a chemical agent (e.g., dithiothreitol), or any combinationthereof. In some embodiments, the cleavable linker can be sensitive tolight and enzyme degradation.

In some embodiments where a photocleavable linker is used, theidentification nucleotide sequences can be released from the boundtarget probes by exposing the bound target probes to a light of aspecified wavelength. In some embodiments, ultraviolet light can be usedto release identification nucleotide sequences from bound target probes.

The signals from the released identification nucleotide sequences can bedetected by various methods known in the art, including, but not limitedto sequencing, quantitative polymerase chain reaction (PCR), multiplexed(PCR), mass cytometry, fluorophore-inactivated multiplexedimmunofluorescence, hybridization-based methods, fluorescencehybridization-based methods, imaging, and any combinations thereof. Insome embodiments, the signals from the released identificationnucleotide sequences can be determined by electrophoresis-based methods.In some embodiments, the signals from the released identificationnucleotide sequences are not determined by electrophoresis-basedmethods. Gel electrophoresis-based methods are generally not asquantitative or sensitive as other detection methods described hereinsuch as PCR, fluorescence hybridization-based methods, and nanoStringnCounter® hybridization technology, for example, as described in U.S.Pat. No. 8,415,102, and Geiss et al. Nature Biotechnology. 2008. 26(3):317-325, the contents of each of which is incorporated herein byreference. Thus, gel electrophoresis-based methods do not necessarilyhave required sensitivity for detection of rare proteins, e.g., proteinsthat are not generally highly-expressed, or not easily accessible orextracted, such as intracellular proteins. In addition, limited sizeresolution on gels can limit simultaneous measurements of a large number(e.g., more than 5 or more than 10) of different target molecules, ascompared to other detection methods described herein such as PCR,fluorescence hybridization-based methods, and nanoString nCounter®hybridization technology, for example, as described in U.S. Pat. No.8,415,102, and Geiss et al. Nature Biotechnology. 2008. 26(3): 317-325,the contents of each of which is incorporated herein by reference.

The nature of the signals from the released identification nucleotidesequences can vary with choice of detection methods and/or detectablelabels. In some embodiments, the signals from the releasedidentification nucleotide sequences can be detected byhybridization-based methods. For example, in some embodiments, themethod can further comprise, prior to detecting the signals from thereleased identification nucleotide sequences, coupling the releasedidentification nucleotide sequences to a detection compositioncomprising a plurality of reporter probes. Each reporter probe in theplurality can comprise (i) a first target probe-specific region that iscapable of binding a first portion of the identification nucleotidesequence; and (ii) a detectable label that identifies the reporterprobe. In these embodiments, signals from the respective detectablelabels of the reporter probes that are coupled to the releasedidentification nucleotide sequences can be detected accordingly. Sincethe signals are distinguishable for each respective reporter probes thatare bound to the identification nucleotide sequences, target-bindingmolecules can be correspondingly identified, thereby detecting aplurality of target molecules in the sample. Additional information of“reporter probes” will be found in the following “Reporter Probes”section.

In some embodiments, the detection composition used in the methodsdescribed herein can additionally comprise a plurality of capture probesas described herein. Additional information of capture probes will befound in the “Capture Probes” section below.

In some embodiments, the method selected to detect signals from thereleased identification nucleotide sequences does not requireamplification of the released identification nucleotide sequences, firsttarget probe-specific region, or the second target probe-specificregion. Amplification-free detection methods can minimize any bias orerrors introduced during amplification, e.g., due to varyingamplification efficiencies among the nucleotide sequences.

In some embodiments, the identification nucleotide sequences can bedetected by nanoString nCounter® hybridization technology, for example,as described in U.S. Pat. No. 8,415,102, and Geiss et al. NatureBiotechnology. 2008. 26(3): 317-325, the contents of each of which isincorporated herein by reference.

Typically, signals detected from the identification nucleotide sequencesof the target probes corresponding to target molecules can be comparedto a control reference to account for any non-specific binding.Accordingly, in some embodiments, the composition added to the samplecan further comprise a plurality of control probes. Each control probein the plurality can comprise: (i) a control-binding molecule thatspecifically binds to one control molecule in the sample; (ii) anidentification control sequence that identifies the control-bindingmolecule; and (iii) a cleavable linker between the control-bindingmolecule and the identification control sequence. The control-bindingmolecule can bind to a control protein present in a sample. Non-limitingexamples of control proteins include housekeeping proteins, control IgGisotypes, mutant non-functional or non-binding proteins, and anycombinations thereof.

Signals from the control probes can then be used to threshold thesignals from the target probes. Accordingly, in some embodiments, themethod can further comprise thresholding the target signals. In someembodiments, the target signals can be thresholded on the basis ofnonspecific binding. For example, in some embodiments, the threshold canbe determined by using standard deviation and measurement error from atleast one or more control proteins. The threshold is generally set to begreater than that of the signals from the non-specific binding. In someembodiments, the threshold can be at least 50% or more (including, e.g.,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orhigher) greater than that of the signals from the non-specific binding.In some embodiments, the threshold can be at least 1.1-fold or more(including, e.g., at least 1.2-fold, at least 1.3-fold, at least1.4-fold, at least 1.5-fold, at least 2-fold, or higher) greater thanthat of the signals from the non-specific binding.

In some embodiments, the method can further comprise quantifying thesignals (e.g., signals that are above the pre-determined threshold) bynormalizing the signals associated with the target probes by the signalsassociated with the control probes. In some embodiments, the signals canbe analyzed and expressed as number of identification nucleotidesequences per target-binding molecule (or target molecule).

In some embodiments, the methods described herein can complement otherart-recognized single-cell proteomic techniques. Exemplary single-cellproteomic techniques include, e.g., mass cytometry andfluorophore-inactivated multiplexed immunofluorescence. See, e.g.,Bendall et al. Science 332, 687-696 (2011) and Gerdes et al. Proc. Natl.Acad. Sci. U.S.A. 110, 11982-11987 (2013) for additional informationabout single-cell mass cytometry and fluorophore-inactivated multiplexedimmunofluorescence.

In some embodiments, the methods, systems and kits described herein canenable measurements of at least two target molecules of different types.For example, the methods, systems, and kits described herein can be usedto measure, for example, nucleic acid molecules and proteins, orproteins and metabolites, or proteins and lipids. The measurements of atleast two target molecules of different types can be performedsimultaneously or sequentially.

By way of example only, the methods, systems and kits described hereinapplied to a sample can preserve genetic materials in a sample whiledetecting other non-genetic target materials in the same sample. This isone of the advantages over existing non-genetic (e.g., proteomic)analysis methods such as flow cytometry and mass cytometry, whichgenerally require an entire cell to measure non-genetic target molecules(e.g., but not limited to proteins). Accordingly, following thenon-genetic (e.g., proteomic) measurements, the entire cell includingits genetic material is lost. In flow cytometry, the cell is lost as itgoes through the flow chamber to detect fluorescence; in mass cytometry,the cellular sample is vaporized, destroying any genetic material thatmay be available. Cell vaporization in mass cytometry results indestruction of ˜60% of the sample even for proteomic detection let alonerecovery genetic material.

In contrast, the methods and/or systems presented herein employ anidentification nucleotide sequence (which comprises nucleotides) as atag or barcode to label and/or measure non-genetic target molecules(e.g., but not limited to proteins). Thus, the methods and/or systemsdescribed herein ensure that any nucleotide-containing materials (e.g.,identification nucleotide sequences and even genetic material extractedfrom cells) will not be destroyed. As such, in one embodiment, themethods to perform simultaneous measurements on the identificationnucleotide sequences (barcodes for identification of non-genetic targetmolecules, e.g., but not limited to proteins) as well as cells' geneticmaterial of interest (including, but not limited to DNA, RNA, microRNAs,long non-coding RNAs, etc.) are essentially the same, except that thecomplementary probe set (comprising reporter probes and optionallycapture probes) has to be expanded to detect not only the identificationnucleotide sequences to measure the non-genetic target molecules (e.g.,but not limited to proteins), but also the genetic materials (e.g., butnot limited to DNA/RNA) from cells.

Accordingly, in some embodiments, the methods, systems and/or kitsdescribed herein for detection of non-genetic target molecules (e.g.,but not limited to proteins) can be used in combination with a nucleiacid analysis for genetic materials, for example, to study thenon-genetic target molecules (e.g., but not limited to proteins) thatinteract with genetic materials or genetic regulatory elements. In theseembodiments, the methods and systems described herein for detecting aplurality of target molecules in a sample as described herein canfurther comprise extracting a nucleic acid molecule from the same samplein which target molecules are to be detected. In some embodiments, themethods and systems described herein can further comprise subjecting theextracted nucleic acid molecule to a nucleic acid analysis. Variousmethods can be used for nucleic acid analysis, including, but notlimited to sequencing, next generation sequencing, quantitativepolymerase chain reaction (PCR), multiplexed (PCR), DNA sequencing, RNAsequencing, de novo sequencing, next-generation sequencing such asmassively parallel signature sequencing (MPSS), polony sequencing,pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ionsemiconductor sequencing, DNA nanoball sequencing, Heliscope singlemolecule sequencing, single molecule real time (SMRT) sequencing,nanopore DNA sequencing, sequencing by hybridization, sequencing withmass spectrometry, microfluidic Sanger sequencing, microscopy-basedsequencing techniques, RNA polymerase (RNAP) sequencing, fluorescencehybridization-based technology (e.g., but not limited to nanoStringnCounter® technology), any art-recognized nucleic acid detectionmethods, or any combinations thereof.

In some embodiments, after a sample and/or non-genetic target moleculeshave been labeled with a plurality of target probes described herein,the identification nucleotide sequences of the target probes can bereleased from the bound non-genetic target molecules simultaneously withextraction of nucleic acid molecules (cells' genetic materials) from thesame labeled sample. In these embodiments, both the nucleic acidmolecules (cells' genetic materials) of interest and the identificationnucleotide sequences can be detected simultaneously in a single samplemixture. In one embodiment, both the nucleic acid molecules (cells'genetic materials) of interest and the identification nucleotidesequences can be detected simultaneously in a single sample mixtureusing nanoString nCounter® analysis system, for example, as described inU.S. Pat. No. 8,415,102, the content of which is incorporated herein byreference. In this embodiment, once the nucleic acid molecules (geneticmaterials) from cells and the released identification nucleotidesequences are in solution, the solution mixture can be contacted withprobe sets comprising both reporter probes and capture probes asdescribed herein for the identification nucleotide sequences as well asfor the cell's nucleic acid molecules (cells' genetic materials) ofinterest. One of the advantages of using nanoString nCounter®hybridization technology is that the analysis can be done on celllysates, as well as on fixed samples, without the need for amplificationthat can introduce bias, and with minimal hands-on time preparation.However, other art-recognized methods for nucleic acid analyses orgenetic analysis as described herein (e.g., but not limited tosequencing) can also be used for simultaneous detection of both nucleicacid molecules (cells' genetic materials) of interest and releasedidentification nucleotide sequences from bound non-genetic targetmolecules. For example, in the case of sequencing, both the cells'genetic materials (e.g., DNA and/or mRNA) and the identificationnucleotide sequences corresponding non-genetic target molecules can besequenced together.

In alternative embodiments, nucleic acid molecules can be extracted froma first portion of a sample, while non-genetic target molecules can beindependently derived or obtained from a second portion of the samesample. In these embodiments, the nucleic acid molecules of interest andthe non-genetic target molecules can be detected separately to determineexpression levels of the nucleic acid molecules of interest andnon-genetic target molecules in the same sample. The nucleic acidmolecules of interests can be subjected to any art-recognized nucleicacid analysis, while the non-genetic target molecules can be detectedthrough detecting and identifying the corresponding identificationnucleotide sequences released from the target probes using the methods,systems and/or kits described herein.

In some embodiments, the methods, systems and/or kits described hereincan be adapted to measure proteins and nucleic acid molecules (cells'genetic materials) present in the same sample. For example, the proteinscan be labeled by one or more embodiments of the target probes describedherein and detected using the methods, systems and/or kits describedherein, while the nucleic acid molecules (cells' genetic materials) canbe detected separately or simultaneously by any methods known in the art(e.g., using, in one embodiment, nanoString nCounter® gene expressionkit), e.g., for a multi-analyte assay on the same sample. In oneembodiment, the sample can comprise cancer cells. The multi-analyteassay can enable generation of an integrated expression profiling forthe sample, which can provide information on interaction between theproteins and the nucleic acid molecules, e.g., genetic regulatoryelements such as microRNAs. This would be valuable or desirable in caseswhere rare samples with only limited sample size are available. Forexample, after labeling a sample or cells or non-genetic targetmolecules (e.g., proteins) with the target probes each comprising anunique identification nucleotide sequence (where in one embodiment, theidentification nucleotide sequences are alien or foreign DNA barcodes),the identification nucleotide sequences (e.g., alien or foreign DNAbarcodes) can then be released from the bound cells or target molecules(e.g., proteins) simultaneously with nucleic acid molecules (e.g., RNAand/or DNA) from the same sample or cells, e.g., using lysis buffer withor without additional cleaving agents (e.g., but not limited to, UVand/or chemical agents). Once the nucleic acid molecules (e.g., RNAand/or DNA) from the cells and unique identification nucleotidesequences (e.g., alien DNA barcodes) are in solution, a hybridizationassay can be performed. In one embodiment, the hybridization assay canbe nanoString nCounter® analysis assay. In the nCounter® analysis assay,the probe sets can have both reporter probes and capture probes asdescribed herein for the identification nucleotide sequences (e.g.,alien DNA barcodes) as well as for the genes of interest. If sample sizeis not a concern, a sample can be aliquoted or split such that theprotein assay and gene expression assay can be run separately to get areadout of both mRNA and protein on the same sample (e.g., a certainpopulation of cells).

In some embodiments, to optimize the nanoString nCounter® hybridizationtechnology for detection of identification nucleotide sequences and/orcells' genetic materials, one can, for example, make sure that all theprobes fall into a linear range when counting them in bulk andexpression of one does not saturate the system. This can readily be donewith optimization by one of skill in the art depending on the kit ofinterest.

In another embodiment, by releasing identification nucleotide sequencesfrom bound target molecules (e.g., proteins), genetic material and theidentification nucleotide sequences can be concurrently extracted from asingle sample, enabling analyses of protein-DNA-RNA interrelationships.

While the methods described herein are described in the context wherethe identification nucleotide sequences are released from bound targetprobes before detection, in some embodiments, the identificationnucleotide sequences do not need to be released from the bound targetprobes. Accordingly, in some embodiments, the methods described hereincan also apply when the identification nucleotide sequences remain boundto target probes during detection.

In certain embodiments, the methods of detection are performed inmultiplex assays, whereby a plurality of target molecules are detectedin the same assay (a single reaction mixture). In a one embodiment, theassay is a hybridization assay in which the plurality of targetmolecules are detected simultaneously. In certain embodiments, theplurality of target molecules detected in the same assay is, at least 2different target molecules, at least 5 different target molecules, atleast 10 different target molecules, at least 20 different targetmolecules, at least 50 different target molecules, at least 75 differenttarget molecules, at least 100 different target molecules, at least 200different target molecules, at least 500 different target molecules, orat least 750 different target molecules, or at least 1000 differenttarget molecules. In other embodiments, the plurality of targetmolecules detected in the same assay is up to 50 different targetmolecules, up to 100 different target molecules, up to 150 differenttarget molecules, up to 200 different target molecules, up to 300different target molecules, up to 500 different target molecules, up to750 different target molecules, up to 1000 different target molecules,up to 2000 different target molecules, or up to 5000 different targetmolecules. In yet other embodiments, the plurality of target moleculesdetected is any range in between the foregoing numbers of differenttarget molecules, such as, but not limited to, from 20 to 50 differenttarget molecules, from 50 to 200 different target molecules, from 100 to1000 different target molecules, or from 500 to 5000 different targetmolecules.

Target Probes

As used herein, the term “target probe” generally refers to a syntheticmolecule that specifically binds to a target molecule for identificationand detection. In accordance with various aspects described herein, eachtarget probe comprises: (i) a target-binding molecule that specificallybinds to a target molecule in a sample; (ii) an identificationnucleotide sequence that identifies the target-binding molecule; and(iii) a cleavable linker between the target-binding molecule and theidentification nucleotide sequence.

In some embodiments, the cleavable linker does not comprise apolynucleotide sequence (e.g., a single-stranded polynucleotidesequence) that is complementary (for basepairing) to at least a portionof the identification nucleotide sequence. That is, in theseembodiments, the identification nucleotide sequence is not released froma target-binding molecule by detaching from the complementarypolynucleotide sequence coupled to the target-binding molecule.Accordingly, in some embodiments, a target probe comprises (i) atarget-binding molecule that specifically binds to the target moleculepresent in the sample; (ii) an identification nucleotide sequence thatidentifies the target-binding molecule; and (iii) a cleavable,non-hybridizable linker between the target-binding molecule and theidentification nucleotide sequence.

Target-binding molecules: A target-binding molecule is a molecule thatspecifically binds to target molecule in a sample. As used herein, theterm “specifically bind(s)” or “specific binding” refers to a targetbinding molecule that binds to a target molecule with a greater affinitythan when it binds to other non-target molecule under the sameconditions. Specific binding is generally indicated by a dissociationconstant of 1 μM or lower, e.g., 500 nM or lower, 400 nM or lower, 300nM or lower, 250 nM or lower, 200 nM or lower, 150 nM or lower, 100 nMor lower, 50 nM or lower, 40 nM or lower, 30 nM or lower, 20 nM orlower, 10 nM or lower, or 1 nM or lower. Typically the nature of theinteraction or binding is noncovalent, e.g., by hydrogen, electrostatic,or van der Waals interactions, however, binding can also be covalent.Target-binding molecules can be naturally-occurring, recombinant orsynthetic. Examples of the target-binding molecule can include, but arenot limited to a nucleic acid, an antibody or a portion thereof, anantibody-like molecule, an enzyme, an antigen, a small molecule, aprotein, a peptide, a peptidomimetic, a carbohydrate, an aptamer, andany combinations thereof. In some embodiments, the target-bindingmolecule does not include a nucleic acid molecule.

In some embodiments, the target-binding molecules can be modified by anymeans known to one of ordinary skill in the art. Methods to modify eachtype of target-binding molecules are well recognized in the art.Depending on the types of target-binding molecules, an exemplarymodification includes, but is not limited to genetic modification,biotinylation, labeling (for detection purposes), chemical modification(e.g., to produce derivatives or fragments of the target-bindingmolecule), and any combinations thereof. In some embodiments, thetarget-binding molecule can be genetically modified. In someembodiments, the target-binding molecule can be biotinylated.

In some embodiments, the target-binding molecule can comprise anantibody or a portion thereof, or an antibody-like molecule. An antibodyor a portion thereof or antibody-like molecule can detect expressionlevel of a cellular protein (including cell surface proteins, secretedproteins, cytoplasmic proteins, and nuclear proteins), orphosphorylation or other post-translation modification state thereof. Insome embodiments, the antibody or a portion thereof or antibody-likemolecule can specifically bind to a protein marker present in a rarecell. Examples of a rare cell include, but are not limited to acirculating tumor cell, a fetal cell, and/or a stem cell. In someembodiments, the antibody or a portion thereof or antibody-like moleculecan specifically bind to a target marker or protein associated with acondition (e.g., a normal healthy state, or a disease or disorder). Insome embodiments, the antibody or a portion thereof or antibody-likemolecule can specifically bind to a target marker or protein associatedwith cancer. For example, target markers or proteins associated withcancer can be involved in apoptosis, epigenetic, DNA damage,kinases/oncogenes, cancer diagnostic markers, epithelial-mesenchymaltransition, autophagy, proliferation, and/or immune response.

In some embodiments, the target-binding molecule can comprise a cellsurface receptor ligand. As used herein, a “cell surface receptorligand” refers to a molecule that can bind to the outer surface of acell. Exemplary cell surface receptor ligand includes, for example, acell surface receptor binding peptide, a cell surface receptor bindingglycopeptide, a cell surface receptor binding protein, a cell surfacereceptor binding glycoprotein, a cell surface receptor binding organiccompound, and a cell surface receptor binding drug. Additional cellsurface receptor ligands include, but are not limited to, cytokines,growth factors, hormones, antibodies, and angiogenic factors.

In some embodiments, the target-binding molecule comprises an antibodyselected from Table 1 in the Example, or a fragment thereof.

In some embodiments, the target-binding molecules of the target probesused in the methods described herein can comprise at least a portion orall of the antibodies listed in Table 1 in the Example, or fragmentsthereof.

Identification nucleotide sequences: As used herein, the term“identification nucleotide sequence” refers to a nucleotide sequencethat identifies a specific target-binding molecule. Thus, eachidentification nucleotide sequence acts as a unique identification codefor each target-binding molecule to which it was coupled.

In some embodiments, the identification nucleotide sequences haveminimal or no secondary structures such as any stable intra-molecularbase-pairing interaction (e.g., hairpins). Without wishing to be boundby theory, in some embodiments, the minimal secondary structure in theidentification nucleotide sequences can provide for better hybridizationbetween a first portion of the identification nucleotide sequence andthe reporter probe, and/or between a second portion of theidentification nucleotide sequence and the capture probe. In addition,the minimal secondary structure in the identification nucleotidesequence can provide for better binding of the target-binding moleculeto the corresponding target molecule. In some embodiments, theidentification nucleotide sequences described herein have no significantintra-molecular pairing at a pre-determined annealing temperature. Thepre-determined annealing temperature can range from about 65° C.-80° C.or from about 70° C.-80° C., or at about 70° C.-75° C.

In some embodiments, identification nucleotide sequences of the targetprobes described herein can be selected or designed such that they donot cross-react with or bind to any nucleic acid sequence in a genome ofa subject whose sample is being evaluated. Thus, the identificationnucleotide sequences of the target probes used to detect targetmolecules in a subject's sample can be selected or designed based onnucleotide sequences of a species or genus that share a homology(sequence identity) with that of the subject by no more than 50% orless, including, e.g., no more than 40%, no more than 30%, no more than20%, no more than 10% or less. In some embodiments, the identificationnucleotide sequences can be pre-screened for no homology against majororganisms (e.g., but not limited to human and/or other mammals) in theNCBI Reference Sequence (RefSeq) database. By way of example only, insome embodiments, the identification nucleotide sequences of the targetprobes used in an animal's sample (e.g., a mammal such as a human) canbe derived from a plant genome. In one embodiment, the identificationnucleotide sequences of the target probes used in a human's sample canbe derived from a potato genome. In some embodiments, the identificationnucleotide sequence can comprise a sequence selected from Table 2 (SEQID NO: 1 to SEQ ID NO: 110), or a fragment thereof.

Generally, identification nucleotide sequences of the target probes canhave any sequence length and can vary depending on a number of factors,including, but not limited to detection methods, and/or the number oftarget molecules to be detected. For example, in some embodiments, thelength of the identification nucleotide sequences can increase toprovide sufficient identification of a large number of target moleculesin a sample. In some embodiments where a hybridization-based method isused to detect identification nucleotide sequences, the identificationnucleotide sequences can have a length sufficient to provide reliablebinding to complementary reporter probes and/or capture probes and togenerate detectable signals. In some embodiments, the identificationnucleotide sequences can have a length of about 30-150 nucleotides, orabout 30-100 nucleotides, or about 50-100 nucleotides. In someembodiments, the identification nucleotide sequences can have a lengthof at least about 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100 or more nucleotides. In someembodiments, the identification nucleotide sequences can have a lengthof about 70 nucleotides.

In some embodiments, the identification nucleotide sequences describedherein can have a fairly consistent melting temperature (Tm). Withoutwishing to be bound by theory, the Tm of the identification nucleotidesequences described herein refers to the temperature at which 50% of theoligonucleotide and its complement are in duplex. The consistent Tmamong a population of the identification nucleotide sequences allows forthe synthesis and hybridization procedures to be tightly optimized, asthe optimal conditions are substantially the same for all spots andpositions. In some embodiments, the Tm of an identification nucleotidesequence when hybridized to its complementary reporter probes and/orcapture probes can be selected to minimize any potential formation ofsecondary structures (e.g., hairpins) that could interfere with probehybridization. In some embodiments, the Tm of an identificationnucleotide sequence when hybridized to its complementary reporter probesand/or capture probes can range from about 70-90° C., from about 75-85°C., or from about 79-82° C. In some embodiments, the Tm of anidentification nucleotide sequence when hybridized to its complementaryreporter probes and/or capture probes can be at least 70° C., at least75° C., at least 80° C., at least 85° C. or higher. In some embodiments,the Tm of an identification nucleotide sequence when hybridized to itscomplementary reporter probes and/or capture probes can be about 80° C.

The GC content of the identification nucleotide sequences can varydepending on a number of factors including, e.g., desired lengths ofreporter probes and/or capture probes described and/or desired Tmtemperatures. For example, when the reporter and/or capture probes areshorter, the GC content of the identification nucleotide sequences canbe increased to maintain the desired Tm consistent between the reporterand/or capture probes and identification nucleotide sequences tominimize potential formation of secondary structures (e.g., hairpins)that could interfere with probe hybridization. In one embodiment, the GCcontent of the identification nucleotide sequence is optimized tomaintain the Tm of an identification nucleotide sequence when hybridizedto its complementary reporter probes and/or capture probes to be about80° C.

In some embodiments, the identification nucleotide sequences have abalanced GC content. For example, in some embodiments, no singlenucleotide in the identification nucleotide sequence can run longer than3 nt. For example, no G nucleotide or C nucleotide runs longer than 3 ntin the identification nucleotide sequence. In one embodiment where thereporter and/or capture probes have a length of about 35 nucleotides,the GC content can be optimized to maintain the Tm of the correspondingidentification nucleotide sequences to be about 80° C.; where theidentification nucleotide sequence should have a balanced GC content asmuch as possible to avoid local regions of very high GC or poly C/poly Gruns.

In some embodiments, the identification nucleotide sequences are DNAsequences.

Cleavable linkers: As used herein, the term “cleavable linker” refers toa linker which is sufficiently stable under one set of conditions, butwhich is cleaved under a different set of conditions to release the twoparts the linker is holding together. In some embodiments, the cleavablelinker can be cleaved at least 1.5 times or more (including, e.g., atleast 2 times, at least 3 times, at least 4 times, at least 5 times, atleast 6 times, at least 7 times, at least 8 times, at least 9 times, atleast 10 times, at least 20 times, at least 30 times, at least 40 times,at least 50 times or more) faster under a first reference condition(e.g., with a cleaving agent) than under a second reference condition(e.g., without a cleaving agent).

For example, a cleavable linker couples an identification nucleotidesequence and a target-binding agent together under one set of conditionsand can be cleaved, digested or degraded under a different set ofconditions to release the identification nucleotide sequence. Thecleavable linker coupling a target-binding molecule to an identificationnucleotide sequence in a target probe can control release of theidentification nucleotide sequence from the target probe when needed,e.g., upon binding to a target molecule, such that the identificationnucleotide can be released for detection. Cleavable linkers are known inthe art, of which examples include, but are not limited to the ones thatare sensitive to an enzyme, pH, temperature, light, shear stress,sonication, a chemical agent (e.g., dithiothreitol), or any combinationthereof. In some embodiments, the cleavable linker can be sensitive tolight and protein degradation, e.g., by an enzyme.

Cleavable linkers are susceptible to cleavage agents, e.g., hydrolysis,pH, redox potential, and light (e.g., infra-red, and/or UV) or thepresence of degradative molecules. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable linker by reduction;esterases; amidases; endosomes or agents that can create an acidicenvironment, e.g., those that result in a pH of five or lower; enzymesthat can hydrolyze or degrade an acid cleavable linker by acting as ageneral acid, peptidases (which can be substrate specific) andproteases, and phosphatases. In some embodiments, the cleavable linkercan be cleavable by a particular enzyme.

In some embodiments, the cleavable linker is a cleavable,non-hybridizable linker. As used herein, the term “cleavable,non-hybridizable linker” refers to a cleavable linker as defined earlierthat does not comprise a polynucleotide sequence (e.g., asingle-stranded polynucleotide sequence) complementary (for basepairing)to at least a portion of the identification nucleotide sequencedescribed herein. That is, in these embodiments, the identificationnucleotide sequence is not released from the target-binding molecule bydetaching from the complementary polynucleotide sequence coupled to thetarget-binding molecule.

Exemplary cleavable, non-hybridizable linkers include, but are notlimited to, hydrolyzable linkers, redox cleavable linkers (e.g., —S—S—and —C(R)₂—S—S—, wherein R is H or C₁-C₆ alkyl and at least one R isC₁-C₆ alkyl such as CH₃ or CH₂CH₃); phosphate-based cleavable linkers(e.g., —O—P(O)(OR)—O—, —O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—,—O—P(O)(OR)—S—, —S—P(O)(OR)—S—, —O—P(S)(OR)—S—, —S—P(S)(OR)—O—,—O—P(O)(R)—O—, —O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—,—S—P(O)(R)—S—, —O—P(S)(R)—S—, . —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, whereinR is optionally substituted linear or branched C1-C10 alkyl); acidcleavable linkers (e.g., hydrazones, esters, and esters of amino acids,—C═NN— and —OC(O)—); ester-based cleavable linkers (e.g., —C(O)O—);peptide-based cleavable linkers, (e.g., linkers that are cleaved byenzymes such as peptidases and proteases in cells, e.g.,—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids), photocleavable linkers and anycombinations thereof. A peptide based cleavable linker comprises two ormore amino acids. In some embodiments, the peptide-based cleavagelinkage comprises the amino acid sequence that is the substrate for apeptidase or a protease. In some embodiments, an acid cleavable linkeris cleavable in an acidic environment with a pH of about 6.5 or lower(e.g., about 6.5, 6.0, 5.5, 5.0, or lower), or by agents such as enzymesthat can act as a general acid.

In some embodiments, the cleavable, non-hybridizable linker can comprisea disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydrylgroup, a nitrobenzyl group, a nitoindoline group, a bromohydroxycoumarin group, a bromo hydroxyquinoline group, a hydroxyphenacylgroup, a dimethozybenzoin group, or any combinations thereof.

In some embodiments, the cleavable, non-hybridizable linker can compriseat least one of the linkers shown in FIG. 2A.

In some embodiments, the cleavable, non-hybridizable linker can comprisea photocleavable linker. A photocleavable linker is a linker that can becleaved by exposure to electromagnetic radiation (e.g., visible light,UV light, infrared, etc.). The wavelength of light necessary tophotocleave the linker is dependent upon the structure of thephotocleavable linker used. Any art-recognized photocleavable linker canbe used for the target probes described herein. Exemplary photocleavablelinker include, but are not limited to, chemical molecules containing ano-nitrobenzyl moiety, a p-nitrobenzyl moiety, a m-nitrobenzyl moiety, anitoindoline moiety, a bromo hydroxycoumarin moiety, a bromohydroxyquinoline moiety, a hydroxyphenacyl moiety, a dimethozybenzoinmoiety, or any combinations thereof.

Additional exemplary photocleavable groups are generally described andreviewed in Pelliccioli et al., Photoremovable protecting groups:reaction mechanisms and applications, Photochem. Photobiol. Sci. 1441-458 (2002); Goeldner and Givens, Dynamic Studies in Biology,Wiley-VCH, Weinheim (2005); Marriott, Methods in Enzymology, Vol. 291,Academic Press, San Diego (1998); Morrison, Bioorganic Photochemistry,Vol. 2, Wiley, New York (1993); Adams and Tsien, Annu. Rev. Physiol. 55755-784 (1993); Mayer et al., Biologically Active Molecules with a“Light Switch,” Angew. Chem. Int. Ed. 45 4900-4921 (2006); Pettit etal., Neuron 19 465-471 (1997); Furuta et al., Brominated7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups withbiologically useful cross-sections for two photon photolysis, Proc.Natl. Acad. Sci. USA 96 1 193-1200 (1999); and U.S. Pat. Nos. 5,430,175;5,635,608; 5,872,243; 5,888,829; 6,043,065; and Zebala, U.S. PatentApplication No. 2010/0105120, the disclosures of which are incorporatedby reference herein.

In some embodiments, the photocleavable linker can generally bedescribed as a chromophore. Examples of chromophores which arephotoresponsive to such wavelengths include, but are not limited to,acridines, nitroaromatics, and arylsulfonamides. The efficiency andwavelength at which the chromophore becomes photoactivated and thusreleases the identification nucleotide sequences described herein willvary depending on the particular functional group(s) attached to thechromophore. For example, when using nitroaromatics, such as derivativesof o-nitrobenzylic compounds, the absorption wavelength can besignificantly lengthened by addition of methoxy groups.

In some embodiments, the photocleavable linker can comprise anitro-aromatic compound. Exemplary photocleavable linkers having anortho-nitro aromatic core scaffold include, but are not limited to,ortho-nitro benzyl (“ONB”), 1-(2-nitrophenyl)ethyl (“NPE”),alpha-carboxy-2-nitrobenzyl (“CNB”), 4,5-dimethoxy-2-nitrobenzyl(“DMNB”), 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (“DMNPE”),5-carboxymethoxy-2-nitrobenzyl (“CMNB”) and((5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl (“CMNCBZ”) photolabilecores. It will be appreciated that the substituents on the aromatic coreare selected to tailor the wavelength of absorption, with electrondonating groups (e.g., methoxy) generally leading to longer wavelengthabsorption. For example, nitrobenzyl (“NB”) and nitrophenylethyl (“NPE”)are modified by addition of two methoxy residues into4,5-dimethoxy-2-nitrobenzyl and 1-(4,5-dimethoxy-2-nitrophenyl)ethyl,respectively, thereby increasing the absorption wavelength range to340-360 nm.

Further, other ortho-nitro aromatic core scaffolds include those thattrap nitroso byproducts in a hetero Diels Alder reaction as generallydiscussed in Zebala, U.S. Patent Application No. 2010/0105120 andPirrung et al., J. Org. Chem. 68: 1 138 (2003). The nitrodibenzofurane(“NDBF”) chromophore offers an extinction coefficient significantlyhigher in the near UV region but it also has a very high quantum yieldfor the deprotection reaction and it is suitable for two-photonactivation (Momotake et al, The nitrodibenzofuran chromophore: a newcaging group for ultra-efficient photolysis in living cells, Nat.Methods 3 35-40 (2006)). The NPP group is an alternative introduced byPfleiderer et al. that yields a less harmful nitrostyryl species(Walbert et al., Photolabile Protecting Groups for Nucleosides:Mechanistic Studies of the 2-(2-Nitrophenyl)ethyl Group, Helv. Chim.Acta 84 1601-161 1 (2001)).

In exemplary embodiments involving UV light, the photocleavable linkerscan be selected from the group consisting of alpha-carboxy-2-nitrobenzyl(CNB, 260 nm), 1-(2-nitrophenyl)ethyl (NPE, 260 nm),4,5-dimethoxy-2-nitrobenzyl (DMNB, 355 nm),1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE, 355 nm),(4,5-dimethoxy-2-nitrobenzoxy)carbonyl (NVOC, 355 nm),5-carboxymethoxy-2-nitrobenzyl (CMNB, 320 nm),((5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl (CMNCBZ, 320 nm),desoxybenzoinyl (desyl, 360 nm), and anthraquino-2-ylmethoxycarbonyl(AQMOC, 350 nm).

Other suitable photocleavable linkers are based on the coumarin system,such as BHC (Furuta and Iwamura, Methods Enzymol. 291 50-63 (1998);Furuta et al., Proc. Natl. Acad. Sci. USA 96 1 193-1200 (1999); Suzukiet al., Org. Lett. 5:4867 (2003); U.S. Pat. No. 6,472,541, thedisclosure of which is incorporated by reference herein). The DMACMlinkage photocleaves in nanoseconds (Hagen et al.,[7-(Dialkylamino)coumarin-4-yl]methyl-Caged Compounds as Ultrafast andEffective Long-Wavelength Phototriggers of 8-Bromo-Substituted CyclicNucleotides, Chem Bio Chem 4 434-442 (2003)) and is cleaved by visiblelight (U.S. patent application Ser. No. 11/402,715 the disclosure ofwhich is incorporated by reference herein). Coumarin-based photolabilelinkages are also available for linking to aldehydes and ketones (Lu etal., Bhc-diol as a photolabile protecting group for aldehydes andketones, Org. Lett. 5 2119-2122 (2003)). Closely related analogues, suchas BHQ, are also suitable (Fedoryak et al., Brominated hydroxyquinolineas a photolabile protecting group with sensitivity to multiphotonexcitation, Org. Lett. 4 3419-3422 (2002)). Another suitablephotocleavable linker comprises the pHP group (Park and Givens, J. Am.Chem. Soc. 119:2453 (1997), Givens et al., New Phototriggers 9:p-Hydroxyphenacyl as a C-Terminal Photoremovable Protecting Group forOligopeptides, J. Am. Chem. Soc. 122 2687-2697 (2000); Zhang et al., J.Am. Chem. Soc. 121 5625-5632, (1999); Conrad et al., J. Am. Chem. Soc.122 9346-9347 (2000); Conrad et al., Org. Lett. 2 1545-1547 (2000)). Aketoprofen derived photolabile linkage is also suitable (Lukeman et al.,Carbanion-Mediated Photocages: Rapid and Efficient Photorelease withAqueous Compatibility, J. Am. Chem. Soc. 127 7698-7699 (2005)).

In some embodiments, a photocleavable linker is one whose covalentattachment to an identification nucleotide sequence and/ortarget-binding agent is reversed (cleaved) by exposure to light of anappropriate wavelength. In some embodiments, release of theidentification nucleotide sequences occurs when the conjugate issubjected to ultraviolet light. For example, photorelease of theidentification nucleotide sequences can occur at a wavelength rangingfrom about 200 to 380 nm (the exact wavelength or wavelength range willdepend on the specific photocleavable linker used, and can be, forexample, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, or 380 or some range therebetween). Insome embodiments, release of the identification nucleotide sequencesoccurs when the conjugate is subjected to visible light. For example,photorelease of the identification nucleotide sequences can occur at awavelength ranging from about 380 to 780 nm (the exact wavelength orwavelength range will depend on the specific photocleavable linker used,and could be, for example, 380, 400, 450, 500, 550, 600, 650, 700, 750,or 780, or some range therebetween). In some embodiments, release of theidentification nucleotide sequences occurs when the conjugate issubjected to infrared light. For example, photorelease of theidentification nucleotide sequences can occur at a wavelength rangingfrom about 780 to 1200 nm (the exact wavelength or wavelength range willdepend on the specific photocleavable linker used, and could be forexample, 780, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200, orsome range therebetween).

In some embodiments, a photocleavable linker can be selected from thegroup consisting of molecules (i)-(xiv) and any combinations thereof,wherein the chemical structures of the molecules (i)-(xiv) are shown asfollows:

where each of the black dots in each molecule represents a connecting orcoupling point that connects, directly or indirectly, to atarget-binding molecule described herein or an identification nucleotidesequence described herein. The connecting point can be a bond, orcomprise an atom, a molecule, and/or a linker described herein. In someembodiments, the connecting point is a bond.

In some embodiments, the photocleavable linker can comprise the molecule(xiv).

In some embodiments, the photocleavable linker is a photocleavablebifunctional linker. In some embodiments, the photocleavable linker is aphotocleavable multi-functional linker.

In some embodiments where a photocleavable linker is used, theidentification nucleotide sequences can be released from the boundtarget probes by exposing the bound target probes to a light of aspecified wavelength. In some embodiments, ultraviolet (UV) light ornear UV light can be used to release identification nucleotide sequencesfrom bound target probes. In some embodiments, release of theidentification nucleotide sequences can occur at a wavelength rangingfrom about 200 nm to about 450 nm.

Activation agents can be used to activate the components to beconjugated together (e.g., identification nucleotide sequences and/ortarget-binding molecules). Without limitations, any process and/orreagent known in the art for conjugation activation can be used.Exemplary surface activation method or reagents include, but are notlimited to, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC or EDAC), hydroxybenzotriazole (HOBT), N-Hydroxysuccinimide (NHS),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate methanaminium (HATU), silanization,sulfosuccinimidyl 6-[3′(2-[pyridyldithio)-propionamido] hexanoate(sulfo-LC-SPDP), 2-iminothiolane (Traut's agent), trans-cycloocteneN-hydroxy-succinimidyl ester (TCO-NHS), surface activation throughplasma treatment, and the like.

Again, without limitations, any art known reactive group can be used forcoupling a photocleavable linker between an identification nucleotidesequence and a target-binding molecule. For example, various surfacereactive groups can be used for surface coupling including, but notlimited to, alkyl halide, aldehyde, amino, bromo or iodoacetyl,carboxyl, hydroxyl, epoxy, ester, silane, thiol, and the like.

Control Probes

As used herein, the term “control probe” generally refers to a syntheticmolecule that specifically binds to a control molecule foridentification and detection. In accordance with various aspectsdescribed herein, each control probe comprises: (i) a control-bindingmolecule that specifically binds to a control molecule in a sample; (ii)an identification control sequence that identifies the control-bindingmolecule; and (iii) a cleavable linker between the control-bindingmolecule and the identification control sequence.

Control-binding molecules: A control-binding molecule is a molecule thatspecifically binds to a control molecule in a sample. Examples of acontrol protein include, but are not limited to, housekeeping proteins(e.g., GAPDH, actin and/or tubulin), control IgG isotypes, mutantnon-functional or non-binding proteins (e.g., nonfunctional ornon-binding antibodies, or mutated proteins such as a protein G that hasbeen mutated at the binding site), and any combinations thereof.Typically the nature of the interaction or binding is noncovalent, e.g.,by hydrogen, electrostatic, or van der Waals interactions, however,binding can also be covalent. Control-binding molecules can benaturally-occurring, recombinant or synthetic. Examples of thecontrol-binding molecule can include, but are not limited to a nucleicacid, an antibody or a portion thereof, an antibody-like molecule, anenzyme, an antigen, a small molecule, a protein, a peptide, apeptidomimetic, a carbohydrate, an aptamer, and any combinationsthereof. In some embodiments, the control-binding molecule does notinclude a nucleic acid molecule.

In some embodiments, the control-binding molecules can be modified byany means known to one of ordinary skill in the art. Methods to modifyeach type of control-binding molecules are well recognized in the art.Depending on the types of control-binding molecules, an exemplarymodification includes, but is not limited to genetic modification,biotinylation, labeling (for detection purposes), chemical modification(e.g., to produce derivatives or fragments of the control-bindingmolecule), and any combinations thereof. In some embodiments, thecontrol-binding molecule can be genetically modified. In someembodiments, the control-binding molecule can be biotinylated.

In some embodiments, the control-binding molecule can comprise anantibody or a portion thereof, or an antibody-like molecule. An antibodyor a portion thereof or antibody-like molecule can detect expressionlevel of a housekeeping protein, e.g., but not limited to GAPDH, actin,and/or tubulin. In some embodiments, the antibody or a portion thereofor antibody-like molecule can specifically bind to a control IgGisotype. In some embodiments, the antibody or a portion thereof orantibody-like molecule can specifically bind to a mutant non-function ornon-binding protein, e.g., a protein G that has been mutated at thebinding site.

Identification control sequences: As used herein, the term“identification control sequence” refers to a nucleotide sequence thatidentifies a specific control-binding molecule. Thus, eachidentification control sequence acts as a unique identification code foreach control-binding molecule to which it was coupled.

In some embodiments, the identification control sequences have minimalor no secondary structures such as any stable intra-molecularbase-pairing interaction (e.g., hairpins). Without wishing to be boundby theory, in some embodiments, the minimal secondary structure in theidentification control sequences can provide for better hybridizationbetween a first portion of the identification control sequence and thereporter probe, and/or between a second portion of the identificationcontrol sequence and the capture probe. In addition, the minimalsecondary structure in the identification control sequence can providefor better binding of the control-binding molecule to the correspondingcontrol molecule. In some embodiments, the identification controlsequences described herein have no significant intra-molecular pairingat a pre-determined annealing temperature. The pre-determined annealingtemperature can range from about 65° C.-80° C. or from about 70° C.-80°C., or at about 70° C.-75° C.

In some embodiments, identification control sequences of the controlprobes described herein can be selected or designed such that they donot cross-react with or bind to any nucleic acid sequence in a genome ofa subject whose sample is being evaluated. Thus, the identificationcontrol sequences of the control probes used to detect control moleculesin a subject's sample can be selected or designed based on nucleotidesequences of a species or genus that share a homology (sequenceidentity) with that of the subject by no more than 50% or less,including, e.g., no more than 40%, no more than 30%, no more than 20%,no more than 10% or less. By way of example only, in some embodiments,the identification control sequences of the control probes used in ananimal's sample (e.g., a mammal such as a human) can be derived from aplant genome. In one embodiment, the identification control sequences ofthe control probes used in a human's sample can be derived from a potatogenome. In some embodiments, the identification control sequence cancomprise a sequence selected from Table 2 (SEQ ID NO: 1 to SEQ ID NO:110), or a fragment thereof.

Generally, identification control sequences of the control probes canhave any sequence length and can vary depending on a number of factors,including, but not limited to detection methods, and/or the number ofcontrol molecules to be detected. For example, in some embodiments, thelength of the identification control sequences can increase to providesufficient identification of a large number of control molecules in asample. In some embodiments where a hybridization-based method is usedto detect identification control sequences, the identification controlsequences can have a length sufficient to provide reliable binding tocomplementary reporter probes and/or capture probes and to generatedetectable signals. In some embodiments, the identification controlsequences can have a length of about 30-150 nucleotides, or about 30-100nucleotides, or about 50-100 nucleotides. In some embodiments, theidentification control sequences can have a length of at least about 30,at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100 or more nucleotides. In some embodiments, theidentification control sequences can have a length of about 70nucleotides.

In some embodiments, the identification control sequences describedherein can have a fairly consistent melting temperature (Tm). Withoutwishing to be bound by theory, the Tm of the identification controlsequences described herein refers to the temperature at which 50% of theoligonucleotide and its complement are in duplex. The consistent Tmamong a population of the identification control sequences allows forthe synthesis and hybridization procedures to be tightly optimized, asthe optimal conditions are substantially the same for all spots andpositions. In some embodiments, the Tm of an identification controlsequence when hybridized to its complementary reporter probes and/orcapture probes can range from about 70-90° C., from about 75-85° C., orfrom about 79-82° C. In some embodiments, the Tm of an identificationcontrol sequence when hybridized to its complementary reporter probesand/or capture probes can be at least 70° C., at least 75° C., at least80° C., at least 85° C. or higher.

Cleavable linkers: Any cleavable linkers used in the target probes canbe used in the control probes. In some embodiments, the cleavable linkercomprises a photocleavable linker. In some embodiments, thephotocleavable linker can be selected from the group consisting ofmolecules (i)-(xiv) shown herein and any combinations thereof. In someembodiments, the photocleavable linker can comprise the molecule (xiv).

Reporter Probes

As used herein, the term “reporter probe” generally refers to asynthetic molecule that binds a first portion of the identificationnucleotide sequence of a target probe and generates a detectable signalthat is distinguishable for the reporter probe and the boundidentification nucleotide sequence.

In some embodiments, the reporter probes have minimal or no secondarystructures such as any stable intra-molecular base-pairing interaction(e.g., hairpins). Without wishing to be bound by theory, the minimalsecondary structure in the reporter probes can provide for betterhybridization between the reporter probe's backbone and a portion of theidentification nucleotide sequences. In addition, the minimal secondarystructure in the reporter probes can provide for better detection of thedetectable label in the reporter probes. In some embodiments, thereporter probes described herein have no significant intra-molecularpairing at a pre-determined annealing temperature. The pre-determinedannealing temperature can range from about 65° C.-80° C. or from about70° C.-80° C., or at about 70° C.-75° C. Secondary structures can bepredicted by programs known in the art such as MFOLD.

In various aspects described herein, a reporter probe generallycomprises a detectable label that identifies the reporter probe. As usedherein, the term “detectable label” refers to a composition capable ofproducing a detectable signal indicative of the presence of a target,e.g., a reporter probe bound to an identification nucleotide sequence ofa target probe. Detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable detectable labels can includefluorescent molecules, radioisotopes, nucleotide chromophores, enzymes,substrates, chemiluminescent moieties, bioluminescent moieties, and thelike. As such, a detectable label is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means needed for the methods and devices describedherein.

In some embodiments, the detectable label of the reporter probes cancomprise one or more labeling molecules that create a unique signal foreach reporter probe. In some embodiments, the detectable label of thereporter probes can comprise one labeling molecule. In some embodiments,the detectable label of the reporter probes can comprise at least two ormore (e.g., at least 2, at least 3, at least 4, at least 5, at least 6or more) labeling molecules. As used herein, the term “labelingmolecule” is a molecule that is capable of producing a detectablesignal, which constitutes at least part of the detectable signalproduced by the detectable label. Accordingly, a labeling molecule canbe a fluorescent molecule, a radioisotope, a nucleotide chromophore, anenzyme, a substrate, a chemiluminescent moiety, a bioluminescent moiety,or any combinations thereof.

In some embodiments, the detectable label and/or labeling molecule(s)can generate an optical signal. The optical signal can be alight-emitting signal or a series or sequence of light-emitting signals.In some embodiments, labeling molecules for generation of an opticalsignal can comprise one or a plurality of (e.g., at least 2 or more,including, e.g., at least 3, at least 4, at least 5 or more) afluorochrome moiety, a fluorescent moiety, a dye moiety, achemiluminescent moiety, or any combinations thereof.

A wide variety of fluorescent reporter dyes are known in the art.Typically, the fluorophore is an aromatic or heteroaromatic compound andcan be a pyrene, anthracene, naphthalene, acridine, stilbene, indole,benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine,salicylate, anthranilate, coumarin, fluorescein, rhodamine or other likecompound.

Exemplary fluorophores include, but are not limited to, 1,5 IAEDANS;1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10);5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein);5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); Alexa Fluor350™; Alexa Fluor430™; AlexaFluor488™; Alexa Fluor 532™; Alexa Fluor546™; Alexa Fluor568™; AlexaFluor594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor660™; AlexaFluor680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC,AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D;Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTS; AstrazonBrilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G;Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF(low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP(Y66H); BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV;BOBO™-1; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570;Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X;Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6GSE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR;Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; BrilliantSulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green;Calcium Green-1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+;Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CFDA; CFP—Cyan Fluorescent Protein; Chlorophyll;Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPMMethylcoumarin; CTC; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™;Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); DIDS;Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97;Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1);Euchrysin; Europium (III) chloride; Europium; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4;Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UVexcitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv;Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS;Lissamine Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; LuciferYellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; MagnesiumOrange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF;Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; MitotrackerGreen FM; Mitotracker Orange; Mitotracker Red; Mitramycin;Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS(Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red;Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow;Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green 488-X; OregonGreen™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue;Pararosaniline (Feulgen); PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5;PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; PhorwiteBKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C; S65L;S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™;sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SPQ(6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine G Extra; Tetracycline; Tetramethylrhodamine;Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); ThiazineRed R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC(TetramethylRodamineIsoThioCyanate); True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66 W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1;and YOYO-3. Many suitable forms of these fluorescent compounds areavailable and can be used.

Other exemplary detectable labels and/or labeling molecules includeluminescent and bioluminescent markers (e.g., biotin, luciferase (e.g.,bacterial, firefly, click beetle and the like), luciferin, andaequorin), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes(e.g., galactosidases, glucorinidases, phosphatases (e.g., alkalinephosphatase), peroxidases (e.g., horseradish peroxidase), andcholinesterases), and calorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, and latex)beads. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and4,366,241, each of which is incorporated herein by reference.

Means of detecting such detectable labels and/or labeling molecules arewell known to those of skill in the art. Thus, for example, radiolabelscan be detected using photographic film or scintillation counters,fluorescent markers can be detected using a photo-detector to detectemitted light. Enzymatic labels are typically detected by providing theenzyme with an enzyme substrate and detecting the reaction productproduced by the action of the enzyme on the enzyme substrate, andcalorimetric labels can be detected by visualizing the colored label.

In some embodiments, the detectable label and/or labeling molecules cancomprise at least one or more (e.g., at least two, at least three, atleast four, at least five, at least six, at least or seven or more)fluorophores or quantum dots. Without wishing to be bound by a theory,using a fluorescent reagent can reduce signal-to-noise in theimaging/readout, thus maintaining sensitivity. The color sequence of thelabeling molecules in the detectable label can provide an identity tothe corresponding reporter probe. For example, a reporter probe Icomprises a detectable label with three fluorophores in the followingorder: fluorophore A; fluorophore B and fluorophore C; whereas areporter probe II comprises a detectable label with the same threefluorophores but in a different order: fluorophore A; fluorophore C andfluorophore B. While the reporter probes I and II have the samefluorophores, the color sequences of the reporter probe I and reporterprobe II are distinct, which identifies the individual reporter probes.

In some embodiments, the labeling molecule can comprise an enzyme thatproduces a change in color of an enzyme substrate. A variety of enzymessuch as horseradish peroxidase (HRP) and alkaline peroxide (AP) can beused, with either colorimetric or fluorogenic substrates. In someembodiments, the reporter-enzyme produces a calorimetric change whichcan be measured as light absorption at a particular wavelength.Exemplary enzymes include, but are not limited to, beta-galactosidases,peroxidases, catalases, alkaline phosphatases, and the like.

In some embodiments, the reporter probe can further comprise a firsttarget probe-specific region that binds to a first portion of theidentification nucleotide sequence of a target probe. Accordingly, insome embodiments, a reporter probe can comprise: (a) a first targetprobe-specific region that binds to a first portion of theidentification nucleotide sequence; and (b) a detectable label thatidentifies the reporter probe.

As used herein, the term “first target probe-specific region” refers toa region of a reporter probe that binds to a first portion of theidentification nucleotide sequence of a target probe. The first targetprobe-specific region can comprise a fairly regularly-spaced pattern ofa nucleotide residue and/or a group (e.g., at least 2 or more) ofnucleotide residues in the backbone. In some embodiments, a nucleotideresidue and/or a group (e.g., at least 2 or more) of nucleotide residuescan be spaced at least an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 4, 15, 20, 25, 30, 35, 40, 45, or 50 bases apart within thefirst target probe-specific region. This allows for a first targetprobe-specific region having a regularly spaced pattern of a nucleotideor a group of nucleotides binds to a nucleotide sequence that has acomplementary nucleotide or a group of complementary nucleotidesregularly spaced apart by about the same number of bases. For example,in some embodiments, when the first target probe-specific region containa fairly regularly-spaced pattern of adenine residues in the backbone,it can bind a nucleotide sequence that has a thymine residue fairlyregularly spaced apart by about the same of number of bases.

In some embodiments, at least 30% or more (including, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 97%, at least 99%, or 100%) of the firsttarget probe-specific region is complementary to a first portion of theidentification nucleotide sequence. As used herein and throughout thespecification, the term “complementary” refers to a first nucleic acidstrand able to form hydrogen bond(s) with a second nucleic acid strandby either traditional Watson-Crick or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” or 100% complementarity means that all thecontiguous residues of a nucleic acid sequence will form hydrogen bondswith the same number of contiguous residues in a second nucleic acidsequence. Less than perfect complementarity refers to the situation inwhich some, but not all, nucleotides of two strands can form hydrogenbonds with each other. “Substantial complementarity” refers topolynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be non-complementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric sequence to non-target sequences under conditions inwhich specific binding is desired, i.e., in vitro assays, underconditions in which the assays are performed. The non-target sequencestypically differ by at least 1, 2, 3, 4, or 5 nucleotides.

In some embodiments, the first target probe-specific region can beidentified for use in the reporter probe using the methods and computersystems described in U.S. Pat. No. 8,415,102 to NanoString Technologies,Inc.

In some embodiments, the first target probe-specific region and adetectable label can be coupled to each other by at least one or morelinkers as described herein. In some embodiments, the linker between thefirst target probe-specific region and the detectable label can comprisean amide bond. In some embodiments, the linker between the first targetprobe-specific region and the detectable label can comprise a chemicallinker as described herein.

In some embodiments, the detectable label and/or labeling molecules canbe detected using an epifluorescent microscope. In some embodiments, thedetectable label and/or labeling molecules can be detected using afluorescent microscope.

In some embodiments, the detectable label and/or labeling molecules canbe detected through use of spectroscopy. Numerous types of spectroscopicmethods can be used. Examples of such methods include, but are notlimited to, ultraviolet spectroscopy, visible light spectroscopy,infrared spectroscopy, x-ray spectroscopy, fluorescence spectroscopy,mass spectroscopy, plasmon resonance (e.g., Cherif et al., ClinicalChemistry, 52:255-262 (2006) and U.S. Pat. No. 7,030,989; hereinincorporated by reference), nuclear magnetic resonance spectroscopy,Raman spectroscopy, fluorescence quenching, fluorescence resonanceenergy transfer, intrinsic fluorescence, ligand fluorescence, and thelike.

In some embodiments, the detectable label and/or labeling molecules canbe detected through use of fluorescence anisotropy. Fluorescenceanisotropy is based on measuring the steady state polarization of samplefluorescence imaged in a confocal arrangement. A linearly polarizedlaser excitation source preferentially excites fluorescent targetmolecules with transition moments aligned parallel to the incidentpolarization vector. The resultant fluorescence is collected anddirected into two channels that measure the intensity of thefluorescence polarized both parallel and perpendicular to that of theexcitation beam. With these two measurements, the fluorescenceanisotropy, r, can be determined from the equation: r=(Intensityparallel-Intensity perpendicular)/(Intensity parallel+2(Intensityperpendicular)) where the I terms indicate intensity measurementsparallel and perpendicular to the incident polarization. Fluorescenceanisotropy detection of fluorescent molecules has been described.Accordingly, fluorescence anisotropy can be coupled to numerousfluorescent labels as have been described herein and as have beendescribed in the art.

In some embodiments, the detectable label and/or labeling molecules canbe detected through use of fluorescence resonance energy transfer(FRET). Fluorescence resonance energy transfer refers to an energytransfer mechanism between two fluorescent molecules. A fluorescentdonor is excited at its fluorescence excitation wavelength. This excitedstate is then nonradiatively transferred to a second molecule, thefluorescent acceptor. Fluorescence resonance energy transfer may be usedwithin numerous configurations to detect the detectable label and/orlabeling molecules. For example, in some embodiments, a first labelingmolecule can be labeled with a fluorescent donor and second labelingmolecule can be labeled with a fluorescent acceptor. Accordingly, suchlabeled first and second labeling molecules can be used withincompetition assays to detect the detectable label and/or labelingmolecules. Numerous combinations of fluorescent donors and fluorescentacceptors can be used for detection.

In some embodiments, the detectable and/or labeling molecules can bedetected through use of polynucleotide analysis. Examples of suchmethods include, but are not limited to, those based on polynucleotidehybridization, polynucleotide ligation, polynucleotide amplification,polynucleotide degradation, and the like. Methods that utilizeintercalation dyes, fluorescence resonance energy transfer, capacitivedeoxyribonucleic acid detection, and nucleic acid amplification havebeen described, for example, in U.S. Pat. Nos. 7,118,910 and 6,960,437;herein incorporated by reference). Such methods can be adapted toprovide for detection of the detectable label and/or labeling molecules.In some embodiments, fluorescence quenching, molecular beacons, electrontransfer, electrical conductivity, and the like can be used to analyzepolynucleotide interaction. Such methods are known and have beendescribed, for example, in Jarvius, DNA Tools and Microfluidic Systemsfor Molecular Analysis, Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine 161, ACTA UNIVERSITATISUPSALIENSIS UPPSALA 2006, ISBN: 91-554-6616-8; Singh-Zocchi et al, Proc.Natl. Acad. Sci, 100:7605-7610 (2003); Wang et al. Anal. Chem,75:3941-3945 (2003); and Fan et al, Proc. Natl. Acad. Sci, 100:9134-9137(2003) and in U.S. Pat. Nos. 6,958,216; 5,093,268; and 6,090,545, thecontent of all of which is incorporated herein by reference. In someembodiments, the polynucleotide analysis is by polymerase chain reaction(PCR). The fundamentals of PCR are well-known to the skilled artisan,see, e.g. McPherson, et al., PCR, A Practical Approach, IRL Press,Oxford, Eng. (1991), hereby incorporated by reference.

In some embodiments, the reporter probes can further comprise anaffinity tag, which is described in detail in the “Capture probes”section below.

In some embodiments, an affinity tag is placed near or at one end of thereporter probe such that attachment of the reporter probe to a solidsubstrate surface does not significantly interfere with detection of thedetectable label.

In some embodiments, the reporter probe(s) described herein refers to a“reporter probe” or “labeled nanoreporter probe” or “nanoreporterprobe(s)” as described in the U.S. Pat. No. 8,519,115; and US PatentApp. Pub. Nos. US 2014/0017688; US 2014/0037620; US2013/0017971; US2013/0230851; US 2011/0201515; US 2011/0086774; US 2011/0229888; and US2010/0261026, all of which are assigned to Nanostring Technologies, Inc.and are incorporated herein by reference.

Capture Probes

As used herein, the term “capture probe” generally refers to a syntheticmolecule that binds a second portion of the identification nucleotidesequence of a target probe and optionally comprise an affinity tag. Asused herein, the term “affinity tag” refers to a molecule that permitsreversible or reversible immobilization of the capture probe and boundidentification nucleotide sequence to a solid substrate surface, e.g.,to allow visualization and/or imaging of the bound complex. In someembodiments, immobilization of the released identification nucleotidesequences can provide distinguishable spatial signals that identify thecapture probes coupled to the released identification nucleotidesequences. Examples of a solid substrate include, but are not limitedto, a microfluidic device, a cartridge, a microtiter plate, a tube, amagnetic particle, a scaffold, and an array.

The affinity tag of the capture probe can attach to a solid substratesurface through a covalent or non-covalent interaction. Theimmobilization or attachment of the affinity tag to a solid substratesurface can occur covalently or non-covalently using any of the methodsknown to those of skill in the art. For example, covalent immobilizationcan be accomplished through, for example, silane coupling. See, e.g.,Weetall, 15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol.134 (1976). The covalent interaction between the affinity tag and thesolid substrate surface can also be mediated by other art-recognizedchemical reactions, such as NHS reaction or a conjugation agent. Thenon-covalent interaction between the affinity tag and the solidsubstrate surface can be formed based on ionic interactions, van derWaals interactions, dipole-dipole interactions, hydrogen bonds,electrostatic interactions, and/or shape recognition interactions.

In some embodiments, the affinity tag can comprise a linker as describedherein. For example, in some embodiments, the affinity tag can comprisea member of a coupling molecule pair as described in the “Linkers”section below. In some embodiments, the affinity tag can comprise amember of the biotin-avidin or biotin-streptavidin coupling pair. Forexample, in some embodiments, the affinity tag can comprise a biotinmolecule, while the solid surface can be coupled with avidin and/orstreptavidin.

In some embodiments, the affinity tag can comprise a physical linker.For example, the affinity tag can comprise a magnetic particle, wherethe affinity tag is immobilized to a solid substrate surface under amagnetic force.

In some embodiments, the capture probes have minimal or no secondarystructures such as any stable intra-molecular base-pairing interaction(e.g., hairpins). Without wishing to be bound by theory, the minimalsecondary structure in the capture probes can provide for betterhybridization between the capture probe's backbone and a portion of theidentification nucleotide sequences. In addition, the minimal secondarystructure in the capture probes can provide for better attachment of thebound complex (i.e., a complex comprising a capture probe bound to anidentification nucleotide sequence) to a solid substrate surface. Insome embodiments, the capture probes described herein have nosignificant intra-molecular pairing at a pre-determined annealingtemperature. The pre-determined annealing temperature can range fromabout 65° C.-80° C. or from about 70° C.-80° C., or at about 70° C.-75°C.

In some embodiments, the capture probe can comprise a second targetprobe-specific region that binds to a second portion of theidentification nucleotide sequence of a target probe. Accordingly, insome embodiments, a capture probe can comprise: (a) a second targetprobe-specific region that binds to a second portion of theidentification nucleotide sequence; and optionally (b) an affinity tag.

As used herein, the term “second target probe-specific region” refers toa region of a capture probe that binds to a second portion of theidentification nucleotide sequence of a target probe. The second targetprobe-specific region can comprise a fairly regularly-spaced pattern ofa nucleotide residue and/or a group (e.g., at least 2 or more) ofnucleotide residues in the backbone. In some embodiments, a nucleotideresidue and/or a group (e.g., at least 2 or more) of nucleotide residuescan be spaced at least an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 4, 15, 20, 25, 30, 35, 40, 45, or 50 bases apart within thesecond target probe-specific region. This allows for a second targetprobe-specific region having a regularly spaced pattern of a nucleotideor a group of nucleotides binds to a nucleotide sequence that has acomplementary nucleotide or a group of complementary nucleotidesregularly spaced apart by about the same number of bases. For example,in some embodiments, when the second target probe-specific regioncontain a fairly regularly-spaced pattern of adenine residues in thebackbone, it can bind a nucleotide sequence that has a thymine residuefairly regularly spaced apart by about the same of number of bases.

In some embodiments, at least 30% or more (including, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 97%, at least 99%, or 100%) of the secondtarget probe-specific region is complementary to a second portion of theidentification nucleotide sequence.

In some embodiments, the second target probe-specific region can beidentified for use in the capture probe using the methods and computersystems described in U.S. Pat. No. 8,415,102 to NanoString Technologies,Inc.

In some embodiments, the second target probe-specific region and anaffinity tag can be coupled to each other by at least one or morelinkers as described herein. In some embodiments, the linker between thesecond target probe-specific region and the affinity tag can comprise anamide bond. In some embodiments, the linker between the second targetprobe-specific region and the affinity tag can comprise a chemicallinker as described herein.

In some embodiments, the capture probe(s) described herein refers to a“capture probe” or “unlabeled nanoreporter probe” or “nanoreporterprobe(s)” as described in the U.S. Pat. No. 8,519,115; and US PatentApp. Pub. Nos. US 2014/0017688; US 2014/0037620; US2013/0017971; US2013/0230851; US 2011/0201515; US 2011/0086774; US 2011/0229888; and US2010/0261026, all of which are assigned to Nanostring Technologies, Inc.and are incorporated herein by reference.

Where both reporter probes and capture probes are used in the methodsand/or systems described herein, the first target probe-specific regionof a reporter probe and the second target probe-specific region of acapture probe should hybridize to a portion of an identificationnucleotide sequence at different positions. For example, the portions ofthe identification nucleotide sequences to which the target-specificregions of the reporter and capture probes hybridize can be at least 1,at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 30, at least 40 or more base pairsapart.

Systems, e.g., for Multiplexed Detection of Target Molecules in a Sample

Various embodiments of the methods described herein can be carried outin one or more functional modules in a system or a computer system asdescribed herein. Accordingly, another provided herein relates to asystem for multiplexed detection of a plurality of target molecules in asample.

FIG. 18A depicts a device or a computer system 600 comprising one ormore processors 630 and a memory 650 storing one or more programs 620for execution by the one or more processors 630.

In some embodiments, the device or computer system 600 can furthercomprise a non-transitory computer-readable storage medium 700 storingthe one or more programs 620 for execution by the one or more processors630 of the device or computer system 600.

In some embodiments, the device or computer system 600 can furthercomprise one or more input devices 640, which can be configured to sendor receive information to or from any one from the group consisting of:an external device (not shown), the one or more processors 630, thememory 650, the non-transitory computer-readable storage medium 700, andone or more output devices 660.

In some embodiments, the device or computer system 600 can furthercomprise one or more output devices 660, which can be configured to sendor receive information to or from any one from the group consisting of:an external device (not shown), the one or more processors 630, thememory 650, and the non-transitory computer-readable storage medium 700.

In some embodiments, the device or computer system 600 for multiplexeddetection of target molecules in a sample comprises: one or moreprocessors; and memory to store one or more programs, the one or moreprograms comprising instructions for:

-   -   (a) receiving said at least one test sample comprising a sample        and a plurality of target probes described herein;    -   (b) releasing the identification nucleotide sequences from the        target probes that are bound to target molecules in the sample;    -   (c) detecting signals from the released identification        nucleotide sequences;    -   (d) determining the presence of one or more target molecules in        the sample based on the detected signals by performing the        following:    -   i. identifying the detectable probes of the reporter probes that        correspond to the detected signals;    -   ii. identifying the identification nucleotide sequences of the        target probes that correspond to the detectable probes based on        the first target probe-specific regions of the reporter probes;        and    -   iii. identifying the target-binding molecules that correspond to        the identification nucleotide sequences; and    -   (e) displaying a content based in part on the analysis output        from said analysis module, wherein the content comprises a        signal indicative of the following: (i) the presence of one or        more target molecules in the sample, (ii) the absence of one or        more target molecules in the sample, and/or (iii) expression        levels of one or more target molecules in the sample.

FIG. 18B depicts a device or a system 600 (e.g., a computer system) forobtaining data from at least one test sample obtained from at least onesubject is provided. The system can be used for multiplexed detection oftarget molecules in a sample. The system comprises:

-   -   (a) at least one sample processing module 601 comprising        instructions for        -   receiving said at least one test sample comprising a sample            and a plurality of target probes described herein; and        -   releasing the identification nucleotide sequences from the            target probes that are bound to target molecules in the            sample;    -   (b) a signal detection module 602 comprising instructions for        detecting signals from the released identification nucleotide        sequences;    -   (c) at least one data storage module 604 comprising instructions        for storing the detected signals from (b) and information        associated with identification nucleotide sequences of the        target probes;    -   (d) at least one analysis module 606 comprising instructions for        determining the presence of one or more target molecules in the        sample based on the detected signals; and    -   (e) at least one display module 610 for displaying a content        based in part on the analysis output from said analysis module,        wherein the content comprises a signal indicative of the        following: (i) the presence of one or more target molecules in        the sample, (ii) the absence of one or more target molecules in        the sample, and/or (iii) expression levels of one or more target        molecules in the sample.

In some embodiments, the sample processing module 601 can be adapted forisolating target cells, as single cells or as a population, from thesample. In some embodiments, the sample processing module can comprise amicrofluidic device for magnetic separation of target cells orinterfering cells from a sample using the methods and devices asdescribed in as described in the International Pat. App. No. WO2013/078332, the content of which is incorporated herein by reference.

In some embodiments, the sample processing module 601 can comprise amulti-well plate (e.g., 96-well, 384 wells, or nano- or micro-wells) forsingle-cell analyses.

In some embodiments, the sample processing module 601 can be adapted forextracting nucleic acid molecules from the same sample for nucleic acidanalysis. Techniques for nucleic acid analysis are known in the art andcan be used to assay the test sample to determine nucleic acid or geneexpression measurements, for example, but not limited to, DNAsequencing, RNA sequencing, de novo sequencing, next-generationsequencing such as massively parallel signature sequencing (MPSS),polony sequencing, pyrosequencing, Illumina (Solexa) sequencing, SOLiDsequencing, ion semiconductor sequencing, DNA nanoball sequencing,Heliscope single molecule sequencing, single molecule real time (SMRT)sequencing), nanopore DNA sequencing, sequencing by hybridization,sequencing with mass spectrometry, microfluidic Sanger sequencing,microscopy-based sequencing techniques, RNA polymerase (RNAP)sequencing, or any combinations thereof.

Accordingly, in some embodiments, the system described herein can beused to generate integrate profiling, e.g., expression profiles ofproteins and nucleic acid molecules from the same sample.

In some embodiments, the sample processing module 601 or the signaldetection module 602 can further comprise instructions for contactingthe released identification nucleotide sequences with reporter probesdescribed herein.

In some embodiments, the sample processing module 601 or the signaldetection module 602 can further comprise instructions for contactingthe released identification nucleotide sequences with capture probesdescribed herein.

In some embodiments, the sample processing module 601 or the signaldetection module 602 can further comprise instructions for immobilizingthe released identification nucleotides to a solid substrate through theaffinity tag described herein. Examples of a solid substrate include,but are not limited to a microfluidic device, a cartridge, a tube, amicrotiter plate, a magnetic particle, and any combinations thereof.

In some embodiments, the analysis module 606 can further compriseinstructions for (i) identifying the detectable probes of the reporterprobes that correspond to the detected signals; (ii) identifying theidentification nucleotide sequences of the target probes that correspondto the detectable probes based on the first target probe-specificregions of the reporter probes; and (iii) identifying the target-bindingmolecules that correspond to the identification nucleotide sequences,thereby determining the presence of one or more target molecules in thesample based on the detected signals.

In some embodiments, the analysis module 606 can further compriseinstructions for identifying a detectable label corresponding for aplurality of light signals emitted from each detectable label, wherein aspatial or temporal order of the plurality of the light signals isunique for each detectable label.

In some embodiments, the analysis module 606 can further compriseinstructions for thresholding the detected signals. For example, thesignals can be thresholded on the basis of nonspecific binding. In someembodiments, the threshold is greater than that of the signals from thenon-specific binding. By way of example only, the threshold can bedetermined by using standard deviation and measurement error from atleast one control protein. In some embodiments, the threshold can be atleast 50% or more (including, e.g., at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or higher) greater than that of thesignals from the non-specific binding. In some embodiments, thethreshold can be at least 1.1-fold or more (including, e.g., at least1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, atleast 2-fold, or higher) greater than that of the signals from thenon-specific binding.

In some embodiments, the analysis module 606 can further compriseinstructions for quantifying the signals by normalizing the signalsassociated with the target probes by the signals associated with thecontrol probes. In one embodiment, the signals is quantified andexpressed as number of identification nucleotide sequences detected pertarget-binding agent.

Depending on the nature of test samples and/or applications of thesystems as desired by users, the display module 610 can further displayadditional content. In some embodiments where the test sample iscollected or derived from a subject for diagnostic assessment, thecontent displayed on the display module 610 can further comprise asignal indicative of a diagnosis of a condition (e.g., disease ordisorder such as cancer)

In some embodiments wherein the test sample is collected or derived froma subject for selection and/or evaluation of a treatment regimen for asubject, the content can further comprise a signal indicative of atreatment regimen personalized to the subject. In some embodiments, thecontent can further comprise a signal indicative of the treatmentresponse.

A tangible and non-transitory (e.g., no transitory forms of signaltransmission) computer readable medium 700 having computer readableinstructions recorded thereon to define software modules forimplementing a method on a computer is also provided herein. In someembodiments, the computer readable medium 700 stores one or moreprograms for multiplexed detection of target molecules in a sample. Theone or more programs for execution by one or more processors of acomputer system comprises (a) instructions for determining the presenceof one or more target molecules in the sample based on the detectedsignals from the released identification nucleotide sequences byperforming the following: (i) identifying the detectable probes of thereporter probes that correspond to the detected signals; (ii)identifying the identification nucleotide sequences of the target probesthat correspond to the detectable probes based on the first targetprobe-specific regions of the reporter probes; and (iii) identifying thetarget-binding molecules that correspond to the identificationnucleotide sequences; and (b) instructions for displaying a contentbased in part on the analysis output from said analysis module, whereinthe content comprises a signal indicative of the following: (i) thepresence of one or more target molecules in the sample, (ii) the absenceof one or more target molecules in the sample, and/or (iii) expressionlevels of one or more target molecules in the sample.

Depending on the nature of test samples and/or applications of thesystems as desired by users, the computer readable storage medium 700can further comprise instructions for displaying additional content. Insome embodiments where the test sample is collected or derived from asubject for diagnostic assessment, the content displayed on the displaymodule can further comprise a signal indicative of a diagnosis of acondition (e.g., disease or disorder) in the subject. In someembodiments wherein the test sample is collected or derived from asubject for selection and/or evaluation of a treatment regimen for asubject, the content can further comprise a signal indicative of atreatment regimen personalized to the subject. In some embodiments, thecontent can further comprise a signal indicative of the treatmentresponse.

Embodiments of the systems described herein have been described throughfunctional modules, which are defined by computer executableinstructions recorded on computer readable media and which cause acomputer to perform method steps when executed. The modules have beensegregated by function for the sake of clarity. However, it should beunderstood that the modules need not correspond to discrete blocks ofcode and the described functions can be carried out by the execution ofvarious code portions stored on various media and executed at varioustimes. Furthermore, it should be appreciated that the modules mayperform other functions, thus the modules are not limited to having anyparticular functions or set of functions.

Computing devices typically include a variety of media, which caninclude computer-readable storage media and/or communications media, inwhich these two terms are used herein differently from one another asfollows. Computer-readable storage media or computer readable media(e.g., 700) can be any available tangible media (e.g., tangible storagemedia) that can be accessed by the computer, is typically of anon-transitory nature, and can include both volatile and nonvolatilemedia, removable and non-removable media. By way of example, and notlimitation, computer-readable storage media can be implemented inconnection with any method or technology for storage of information suchas computer-readable instructions, program modules, structured data, orunstructured data. Computer-readable storage media can include, but arenot limited to, RAM (random access memory), ROM (read only memory),EEPROM (erasable programmable read only memory), flash memory or othermemory technology, CD-ROM (compact disc read only memory), DVD (digitalversatile disk) or other optical disk storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or other tangible and/or non-transitory media which can be used to storedesired information. Computer-readable storage media can be accessed byone or more local or remote computing devices, e.g., via accessrequests, queries or other data retrieval protocols, for a variety ofoperations with respect to the information stored by the medium.

On the other hand, communications media typically embodycomputer-readable instructions, data structures, program modules orother structured or unstructured data in a data signal that can betransitory such as a modulated data signal, e.g., a carrier wave orother transport mechanism, and includes any information delivery ortransport media. The term “modulated data signal” or signals refers to asignal that has one or more of its characteristics set or changed insuch a manner as to encode information in one or more signals. By way ofexample, and not limitation, communication media include wired media,such as a wired network or direct-wired connection, and wireless mediasuch as acoustic, radio frequency (RF), infrared and other wirelessmedia.

In some embodiments, the computer readable storage media 700 can includethe “cloud” system, in which a user can store data on a remote server,and later access the data or perform further analysis of the data fromthe remote server.

Computer-readable data embodied on one or more computer-readable media,or computer readable medium 700, may define instructions, for example,as part of one or more programs, that, as a result of being executed bya computer, instruct the computer to perform one or more of thefunctions described herein (e.g., in relation to system 600, or computerreadable medium 700), and/or various embodiments, variations andcombinations thereof. Such instructions may be written in any of aplurality of programming languages, for example, Java, J#, Visual Basic,C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, andthe like, or any of a variety of combinations thereof. Thecomputer-readable media on which such instructions are embodied mayreside on one or more of the components of either of system 600, orcomputer readable medium 700 described herein, may be distributed acrossone or more of such components, and may be in transition there between.

The computer-readable media can be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the assays and/or methods described herein. In addition, itshould be appreciated that the instructions stored on the computerreadable media, or computer-readable medium 700, described above, arenot limited to instructions embodied as part of an application programrunning on a host computer. Rather, the instructions may be embodied asany type of computer code (e.g., software or microcode) that can beemployed to program a computer to implement the assays and/or methodsdescribed herein. The computer executable instructions may be written ina suitable computer language or combination of several languages. Basiccomputational biology methods are known to those of ordinary skill inthe art and are described in, for example, Setubal and Meidanis et al.,Introduction to Computational Biology Methods (PWS Publishing Company,Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods inMolecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the system or computersystem described herein can include a sample processing module, a signaldetection module, a storage device, an analysis module and a displaymodule. The functional modules can be executed on one, or multiple,computers, or by using one, or multiple, computer networks. The signaldetection module 602 can have computer executable instructions to detectsignals from the released identification nucleotide sequences.

In some embodiments, the signal detection module 602 can have computerexecutable instructions to provide sequence information in computerreadable form, e.g., for RNA sequencing. In these embodiments, thesystem can enable simultaneous measurements of target molecules (e.g.,proteins) and nucleic acid molecules from the same sample. For example,the integrated expression profiles of the protein and nucleic acidmolecules can be used to study proteins that interact with geneticregulatory elements such as microRNAs. As used herein, “sequenceinformation” refers to any nucleotide and/or amino acid sequence,including but not limited to full-length nucleotide and/or amino acidsequences, partial nucleotide and/or amino acid sequences, or mutatedsequences. Moreover, information “related to” the sequence informationincludes detection of the presence or absence of a sequence (e.g.,detection of a mutation or deletion), determination of the concentrationof a sequence in the sample (e.g., amino acid sequence expressionlevels, or nucleotide (RNA or DNA) expression levels), and the like. Theterm “sequence information” is intended to include the presence orabsence of post-translational modifications (e.g. phosphorylation,glycosylation, summylation, farnesylation, and the like).

As an example, signal detection modules 602 for determining sequenceinformation may include known systems for automated sequence analysisincluding but not limited to Hitachi FMBIO® and Hitachi FMBIO® IIFluorescent Scanners (available from Hitachi Genetic Systems, Alameda,Calif.); Spectrumedix® SCE 9610 Fully Automated 96-CapillaryElectrophoresis Genetic Analysis Systems (available from SpectruMedixLLC, State College, Pa.); ABI PRISM® 377 DNA Sequencer, ABI® 373 DNASequencer, ABI PRISM® 310 Genetic Analyzer, ABI PRISM® 3100 GeneticAnalyzer, and ABI PRISM® 3700 DNA Analyzer (available from AppliedBiosystems, Foster City, Calif.); Molecular Dynamics Fluorlmager™ 575,SI Fluorescent Scanners, and Molecular Dynamics Fluorlmager™ 595Fluorescent Scanners (available from Amersham Biosciences UK Limited,Little Chalfont, Buckinghamshire, England); GenomyxSC™ DNA SequencingSystem (available from Genomyx Corporation (Foster City, Calif.); andPharmacia ALF™ DNA Sequencer and Pharmacia ALFexpress™ (available fromAmersham Biosciences UK Limited, Little Chalfont, Buckinghamshire,England).

Alternative methods for determining sequence information, i.e. signaldetection modules 602, include systems for protein and DNA analysis. Forexample, mass spectrometry systems including Matrix Assisted LaserDesorption Ionization—Time of Flight (MALDI-TOF) systems andSELDI-TOF-MS ProteinChip array profiling systems; systems for analyzinggene expression data (see, for example, published U.S. PatentApplication Pub. No. U.S. 2003/0194711); systems for array basedexpression analysis: e.g., HT array systems and cartridge array systemssuch as GeneChip® AutoLoader, Complete GeneChip® Instrument System,GeneChip® Fluidics Station 450, GeneChip® Hybridization Oven 645,GeneChip® QC Toolbox Software Kit, GeneChip® Scanner 3000 7G plusTargeted Genotyping System, GeneChip® Scanner 3000 7G Whole-GenomeAssociation System, GeneTitan™ Instrument, and GeneChip® Array Station(each available from Affymetrix, Santa Clara, Calif.); automated ELISAsystems (e.g., DSX® or DS2® (available from Dynax, Chantilly, Va.) orthe Triturus® (available from Grifols USA, Los Angeles, Calif.), TheMago® Plus (available from Diamedix Corporation, Miami, Fla.);Densitometers (e.g. X-Rite-508-Spectro Densitometer® (available from RPImaging™, Tucson, Ariz.), The HYRYS™ 2 HIT densitometer (available fromSebia Electrophoresis, Norcross, Ga.); automated Fluorescence in situhybridization systems (see for example, U.S. Pat. No. 6,136,540); 2D gelimaging systems coupled with 2-D imaging software; microplate readers;Fluorescence activated cell sorters (FACS) (e.g. Flow CytometerFACSVantage SE, (available from Becton Dickinson, Franklin Lakes, N.J.);and radio isotope analyzers (e.g. scintillation counters).

The signals from the released identification nucleotide sequencesdetermined in the signal detection module can be read by the storagedevice 604. As used herein the “storage device” 604 is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the system described hereincan include stand-alone computing apparatus, data telecommunicationsnetworks, including local area networks (LAN), wide area networks (WAN),Internet, Intranet, and Extranet, and local and distributed computerprocessing systems. Storage devices 604 also include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage media, magnetic tape, optical storage media such as CD-ROM, DVD,electronic storage media such as RAM, ROM, EPROM, EEPROM and the like,general hard disks and hybrids of these categories such asmagnetic/optical storage media. The storage device 604 is adapted orconfigured for having recorded thereon sequence information orexpression level information. Such information may be provided indigital form that can be transmitted and read electronically, e.g., viathe Internet, on diskette, via USB (universal serial bus) or via anyother suitable mode of communication, e.g., the “cloud.”

As used herein, “expression level information” refers to expressionlevels of any target molecules to be measured, e.g., but not limited to,proteins, peptides, lipids, metabolites, carbohydrates, toxins, growthfactors, hormones, cytokines, cells, and any combinations thereof. Insome embodiments, the expression level information can be determinedfrom the detected signals from the released identification nucleotidesequences corresponding to each target molecule.

As used herein, “stored” refers to a process for encoding information onthe storage device 604. Those skilled in the art can readily adopt anyof the presently known methods for recording information on known mediato generate manufactures comprising the sequence information orexpression level information.

A variety of software programs and formats can be used to store thesequence information or expression level information on the storagedevice. Any number of data processor structuring formats (e.g., textfile or database) can be employed to obtain or create a medium havingrecorded thereon the sequence information or expression levelinformation.

By providing sequence information and/or expression level information incomputer-readable form, one can use the sequence information and/orexpression level information in readable form in the analysis module 606to generate expression profiles for the sample being tested. Theanalysis made in computer-readable form provides a computer readableanalysis result which can be processed by a variety of means. Content608 based on the analysis result can be retrieved from the analysismodule 606 to indicate the presence or absence of one or more targetmolecules present in a sample.

The “analysis module” 606 can use a variety of available softwareprograms and formats for calculating expression profiles of varioustarget molecules. In one embodiment, the analysis module 606 cancalculate proteomic expression profiles as follows. First, raw counts ofthe released identification nucleotide sequences can be first normalizedby using the nSolver analysis software to account for hybridizationdifferences on a cartridge, before normalization via the mean of theinternal positive controls, which account for hybridization efficiency.These counts can then be converted to expression values using therelative counts of identification nucleotide sequences per atarget-binding agent (e.g., an antibody). Next, average backgroundsignal from control IgG can be subtracted. Housekeeping genes can thenbe used for normalization that accounted for cell number variations.Signals can then be normalized via a housekeeping protein, e.g., GAPDH,actin, and/or β-tubulin.

In some embodiments, the analysis module 606 can comprise, e.g., MATLABor functionally equivalent thereof to generate heat maps andclustergrams with a matrix input of marker expression values that werecalculated as described above. In some embodiments, the clustergrams canbe performed as a weighted linkage. In some embodiments, theclustergrams can be clustered using correlation values as a distancemetric. If a target molecule was not detectable, it can be removed fromthe matrix or heat map and is not displayed.

In some embodiments, the analysis module 606 can comprise one or moreprograms for analyzing reporter probes and/or capture probes asdescribed in U.S. Pat. No. 7,941,279 to NanoString Technologies, Inc.

In some embodiments, the analysis module 606 can compare proteinexpression profiles. Any available comparison software can be used,including but not limited to, the Ciphergen Express (CE) and BiomarkerPatterns Software (BPS) package (available from Ciphergen Biosystems,Inc., Freemont, Calif.). Comparative analysis can be done with proteinchip system software (e.g., The Protein chip Suite (available fromBio-Rad Laboratories, Hercules, Calif.). Algorithms for identifyingexpression profiles can include the use of optimization algorithms suchas the mean variance algorithm (e.g. JMP Genomics algorithm availablefrom JMP Software Cary, N.C.).

The analysis module 606, or any other module of the system describedherein, may include an operating system (e.g., UNIX) on which runs arelational database management system, a World Wide Web application, anda World Wide Web server. World Wide Web application includes theexecutable code necessary for generation of database language statements(e.g., Structured Query Language (SQL) statements). Generally, theexecutables will include embedded SQL statements. In addition, the WorldWide Web application may include a configuration file which containspointers and addresses to the various software entities that comprisethe server as well as the various external and internal databases whichmust be accessed to service user requests. The Configuration file alsodirects requests for server resources to the appropriate hardware—as maybe necessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular embodiment, users can directly access data (via Hypertextlinks for example) residing on Internet databases using a HTML interfaceprovided by Web browsers and Web servers. In another embodiment, userscan directly access data residing on the “cloud” provided by the cloudcomputing service providers.

The analysis module 606 provides computer readable analysis result thatcan be processed in computer readable form by predefined criteria, orcriteria defined by a user, to provide a content based in part on theanalysis result that may be stored and output as requested by a userusing a display module 610. The display module 610 enables display of acontent 608 based in part on the comparison result for the user, whereinthe content 608 is a signal indicative of the presence of one or moretarget molecules in the sample, a signal indicative of the absence ofone or more target molecules in the sample, a signal indicative ofexpression levels of one or more target molecules in the sample, or anycombinations thereof. Such signal, can be for example, a display ofcontent 608 on a computer monitor, a printed page of content 608 from aprinter, or a light or sound indicative of the absence of a targetmolecule in a sample.

In various embodiments of the computer system described herein, theanalysis module 606 can be integrated into the signal detection module602.

Depending on the nature of test samples and/or applications of thesystems as desired by users, the content 608 based on the analysisresult can also include a signal indicative of a diagnosis of acondition (e.g., disease or disorder) in the subject. In someembodiments, the content 608 based on the analysis result can furthercomprise a signal indicative of a treatment regimen personalized to thesubject. In some embodiments, the content 608 based on the analysisresult can further comprise a signal indicative of a response of asubject to a treatment, which provides a means of monitoring thetreatment response in a subject.

In some embodiments, the content 608 based on the analysis result caninclude a graphical representation reflecting the expression profiles oftarget molecules, e.g., as shown in FIG. 5.

In one embodiment, the content 608 based on the analysis result isdisplayed a on a computer monitor. In one embodiment, the content 608based on the analysis result is displayed through printable media. Thedisplay module 610 can be any suitable device configured to receive froma computer and display computer readable information to a user.Non-limiting examples include, for example, general-purpose computerssuch as those based on Intel PENTIUM-type processor, Motorola PowerPC,Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety ofprocessors available from Advanced Micro Devices (AMD) of Sunnyvale,Calif., or any other type of processor, visual display devices such asflat panel displays, cathode ray tubes and the like, as well as computerprinters of various types.

In some embodiments, the content can be displayed on a computer display,a screen, a monitor, an email, a text message, a website, a physicalprintout (e.g., paper), or provided as stored information in a datastorage device.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content 608 based on the analysis result.It should be understood that other modules of the system describedherein can be adapted to have a web browser interface. Through the Webbrowser, a user may construct requests for retrieving data from theanalysis module. Thus, the user will typically point and click to userinterface elements such as buttons, pull down menus, scroll bars and thelike conventionally employed in graphical user interfaces. The requestsso formulated with the user's Web browser are transmitted to a Webapplication which formats them to produce a query that can be employedto extract the pertinent information related to the expression profileof target molecules in a sample, e.g., display of an indication of thepresence or absence of one or more target molecules in the sample, ordisplay of information based thereon. In one embodiment, the informationof the control reference is also displayed.

In any embodiments, the analysis module can be executed by a computerimplemented software as discussed earlier. In such embodiments, a resultfrom the analysis module can be displayed on an electronic display. Theresult can be displayed by graphs, numbers, characters or words. Inadditional embodiments, the results from the analysis module can betransmitted from one location to at least one other location. Forexample, the comparison results can be transmitted via any electronicmedia, e.g., internet, fax, phone, a “cloud” system, and anycombinations thereof. Using the “cloud” system, users can store andaccess personal files and data or perform further analysis on a remoteserver rather than physically carrying around a storage medium such as aDVD or thumb drive.

Each of the above identified modules or programs corresponds to a set ofinstructions for performing a function described above. These modulesand programs (i.e., sets of instructions) need not be implemented asseparate software programs, procedures or modules, and thus varioussubsets of these modules may be combined or otherwise re-arranged invarious embodiments. In some embodiments, memory may store a subset ofthe modules and data structures identified above. Furthermore, memorymay store additional modules and data structures not described above.

The illustrated aspects of the disclosure may also be practiced indistributed computing environments where certain tasks are performed byremote processing devices that are linked through a communicationsnetwork. In a distributed computing environment, program modules can belocated in both local and remote memory storage devices.

Moreover, it is to be appreciated that various components describedherein can include electrical circuit(s) that can include components andcircuitry elements of suitable value in order to implement theembodiments of the subject innovation(s). Furthermore, it can beappreciated that many of the various components can be implemented onone or more integrated circuit (IC) chips. For example, in oneembodiment, a set of components can be implemented in a single IC chip.In other embodiments, one or more of respective components arefabricated or implemented on separate IC chips.

What has been described above includes examples of the embodiments ofthe present invention. It is, of course, not possible to describe everyconceivable combination of components or methodologies for purposes ofdescribing the claimed subject matter, but it is to be appreciated thatmany further combinations and permutations of the subject innovation arepossible. Accordingly, the claimed subject matter is intended to embraceall such alterations, modifications, and variations that fall within thespirit and scope of the appended claims. Moreover, the above descriptionof illustrated embodiments of the subject disclosure, including what isdescribed in the Abstract, is not intended to be exhaustive or to limitthe disclosed embodiments to the precise forms disclosed. While specificembodiments and examples are described herein for illustrative purposes,various modifications are possible that are considered within the scopeof such embodiments and examples, as those skilled in the relevant artcan recognize.

In particular and in regard to the various functions performed by theabove described components, devices, circuits, systems and the like, theterms used to describe such components are intended to correspond,unless otherwise indicated, to any component which performs thespecified function of the described component (e.g., a functionalequivalent), even though not structurally equivalent to the disclosedstructure, which performs the function in the herein illustratedexemplary aspects of the claimed subject matter. In this regard, it willalso be recognized that the innovation includes a system as well as acomputer-readable storage medium having computer-executable instructionsfor performing the acts and/or events of the various methods of theclaimed subject matter.

The aforementioned systems/circuits/modules have been described withrespect to interaction between several components/blocks. It can beappreciated that such systems/circuits and components/blocks can includethose components or specified sub-components, some of the specifiedcomponents or sub-components, and/or additional components, andaccording to various permutations and combinations of the foregoing.Sub-components can also be implemented as components communicativelycoupled to other components rather than included within parentcomponents (hierarchical). Additionally, it should be noted that one ormore components may be combined into a single component providingaggregate functionality or divided into several separate sub-components,and any one or more middle layers, such as a management layer, may beprovided to communicatively couple to such sub-components in order toprovide integrated functionality. Any components described herein mayalso interact with one or more other components not specificallydescribed herein but known by those of skill in the art.

In addition, while a particular feature of the subject innovation mayhave been disclosed with respect to only one of several implementations,such feature may be combined with one or more other features of theother implementations as may be desired and advantageous for any givenor particular application. Furthermore, to the extent that the terms“includes,” “including,” “has,” “contains,” variants thereof, and othersimilar words are used in either the detailed description or the claims,these terms are intended to be inclusive in a manner similar to the term“comprising” as an open transition word without precluding anyadditional or other elements.

As used in this application, the terms “component,” “module,” “system,”or the like are generally intended to refer to a computer-relatedentity, either hardware (e.g., a circuit), a combination of hardware andsoftware, software, or an entity related to an operational machine withone or more specific functionalities. For example, a component may be,but is not limited to being, a process running on a processor (e.g.,digital signal processor), a processor, an object, an executable, athread of execution, a program, and/or a computer. By way ofillustration, both an application running on a controller and thecontroller can be a component. One or more components may reside withina process and/or thread of execution and a component may be localized onone computer and/or distributed between two or more computers. Further,a “device” can come in the form of specially designed hardware;generalized hardware made specialized by the execution of softwarethereon that enables the hardware to perform specific function; softwarestored on a computer-readable medium; or a combination thereof.

In view of the exemplary systems described above, methodologies that maybe implemented in accordance with the described subject matter will bebetter appreciated with reference to the flowcharts of the variousfigures. For simplicity of explanation, the methodologies are depictedand described as a series of acts. However, acts in accordance with thisdisclosure can occur in various orders and/or concurrently, and withother acts not presented and described herein. Furthermore, not allillustrated acts may be required to implement the methodologies inaccordance with the disclosed subject matter. In addition, those skilledin the art will understand and appreciate that the methodologies couldalternatively be represented as a series of interrelated states via astate diagram or events. Additionally, it should be appreciated that themethodologies disclosed in this specification are capable of beingstored on an article of manufacture to facilitate transporting andtransferring such methodologies to computing devices. The term articleof manufacture, as used herein, is intended to encompass a computerprogram accessible from any computer-readable device or storage media.

The system 600, and computer readable medium 700, are merelyillustrative embodiments, e.g., for multiplexed detection of targetmolecules in a sample and/or for use in the methods of various aspectsdescribed herein and is not intended to limit the scope of theinventions described herein. Variations of system 600, and computerreadable medium 700, are possible and are intended to fall within thescope of the inventions described herein.

The modules of the machine, or used in the computer readable medium, mayassume numerous configurations. For example, function may be provided ona single machine or distributed over multiple machines.

Kits, e.g., for Multiplexed Detection of Target Molecules in a Sample

Kits, e.g., for multiplexed detection of different target molecules froma sample, are also provided herein. In some embodiments, the kitcomprises (a) a plurality of target probes in accordance with one ormore embodiments described herein; and (b) a plurality of reporterprobes in accordance with one or more embodiments described herein.

In some embodiments, each subset of the target probes in the pluralitybinds to a different target molecule, wherein the target probes in thesubset comprise the same target-binding molecule. That is, no two targetprobes in the subset binds to different regions of the same targetmolecule.

In some embodiments, the kit comprises at least 3 or more (including atleast 4, at least 5, at least 10, at least 15, at least 20, at least 30,at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 150, at least 200, at least 250 ormore) different target probes described herein, wherein each targetprobe specifically binds to a different target molecule. Accordingly, insome embodiments, the kit further comprises at least 3 or more(including at least 4, at least 5, at least 10, at least 15, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 150, at least 200, atleast 250 or more) different reporter probes, wherein each reporterprobe identifies a distinct target probe.

In some embodiments, depending on the design of the identificationnucleotide sequences of the target probes, the kit can further compriseat least one or more (including at least 4, at least 5, at least 10, atleast 15, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 150,at least 200, at least 250 or more) capture probes described herein. Insome embodiments, the same capture probe can be used, e.g., forimmobilization of different identification nucleotide sequences to asolid substrate surface for visualization and/or imaging. In someembodiments, different capture probes can be used, e.g., forimmobilization of different identification nucleotide sequences to asolid substrate surface for visualization and/or imaging.

In some embodiments, reporter probes and capture probes can be providedin the kit individually or in a mixture.

In some embodiments, the target-binding molecules of the target probescan comprise antibodies or fragments thereof. In some embodiments, theantibodies or fragments thereof can be selected from Table 1.

In some embodiments, the cleavable linker of the target probes cancomprise a photocleavable linker. In some embodiments, thephotocleavable linker can be selected from the molecules (i)-(xiv) asshown earlier. In some embodiments, the photocleavable linker cancomprise molecule (xiv).

In some embodiments, the detectable label of the reporter probes cancomprise one or more labeling molecules that create a unique signal foreach reporter probe. An exemplary unique signal can be an opticalsignal. The optical signal can comprise one or a series or a sequence oflight-emitting signals. In these embodiments, non-limiting examples ofthe labeling molecules include fluorochrome moieties, fluorescentmoieties, dye moieties, chemiluminescent moieties, and any combinationsthereof.

In some embodiments, the kit can further comprise a plurality of (e.g.,at least 2 or more, including, at least 3, at least 4, at least 5, atleast 6, at least 7 or more) control probes in accordance with one ormore embodiments described herein.

In some embodiments, the kit can further comprise reagents for detectinga plurality of nucleic acid molecules. Example reagents for nucleic aciddetection and analysis can include, but are not limited to, nucleic acidpolymerase, primers, nucleotides, an agent for nucleic acid extraction,a buffered solution, control nucleic acid sequences, and any combinationthereof. Such kit can be used to generate an integrated profiling thatcombines both target molecule (e.g., protein) and genetic materialinformation (e.g., DNA, RNA, epigenetic, and microRNAs). Thus, the kitcan be used to study target molecules that interact with geneticmaterials such as genetic regulatory elements.

In some embodiments, the kit can further comprise at least one reagentfor use in one or more embodiments of the methods or systems describedherein. Reagents that can be provided in the kit can include at leastone or more of the following: a hybridization reagent, a purificationreagent, an immobilization reagent, an imaging agent, a cellpermeabilization agent, a blocking agent, a cleaving agent for thecleavable linker, and any combinations thereof.

In some embodiments, the kit can further include at least one or moredevices (e.g., sample cartridges or microfluidic devices) or tubes foruse in one or more embodiments of the methods and/or systems describedherein. In some embodiments, the device can comprise a surface forimmobilization of the capture probes upon coupling to the identificationnucleotide sequences. In some embodiments, the device can comprise amicrofluidic device for separating target cells from interfering cellsas described herein. For example, a microfluidic device for magneticseparation of target cells or interfering cells from a sample asdescribed in the International Pat. App. No. WO 2013/078332, the contentof which are incorporated herein by reference, can be included in thekit.

In some embodiments, the kit can further include a computer-readable(non-transitory) storage medium in accordance with one or moreembodiments described herein. For example, in one embodiment, thecomputer-readable (non-transitory) storage medium included in the kitcan provide instructions to determine the presence or expression levelsof one or more target molecules in a sample. The computer-readable(non-transitory) storage medium can be in a CD, DVD, and/or USB drive.

In all such embodiments of the aspect, the kit includes the necessarypackaging materials and informational material therein to store and usesaid kits. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of an agent(s) described herein for the methods describedherein. In one embodiment, the informational material can includeinstructions to perform a multiplexed detection of target molecules in asample. In one embodiment, the information material can includeinstructions to analyze the signal readouts.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In all embodiments of the aspects described herein, the kit willtypically be provided with its various elements included in one package,e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a styrofoambox. The enclosure can be configured so as to maintain a temperaturedifferential between the interior and the exterior, e.g., it can provideinsulating properties to keep the reagents at a preselected temperaturefor a preselected time. The kit can include one or more containers forthe composition containing a compound(s) described herein. In someembodiments, the kit contains separate containers (e.g., two separatecontainers for the two agents), dividers or compartments for thecomposition(s) and informational material. For example, the compositioncan be contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit usage forms of target probes described herein. For example,the kit includes a plurality of syringes, ampules, foil packets, orblister packs, each containing a single unit usage of target probesdescribed herein. The containers of the kits can be air tight,waterproof (e.g., impermeable to changes in moisture or evaporation),and/or light-tight.

Exemplary Uses of the Methods, Systems and Kits Described Herein

The methods, systems and kits described herein can be used in anyapplications where detection of a plurality of target molecules in asample is desirable. For example, a sample can be a biological sample,or a sample from an environmental source (e.g., water, soil, foodproducts, and/or ponds). Other samples that can be analyzed with themethods, systems, and kits described herein are discussed in the“Sample” section below.

The inventors have demonstrated that, in one embodiment, an antibodybarcoding with photocleavable DNA (ABCD) platform described herein canenable analysis of hundreds of proteins from a single cell or a limitednumber of cells, e.g., from minimally invasive fine-needle aspirates(FNAs). Accordingly, samples amenable to the methods described hereincan comprise less than 500 cells or fewer. In some embodiments, thesample can comprise less than 400 cells, less than 300 cells, less than200 cells, less than 100 cells, less than 50 cells, less than 25 cells,less than 5 cells or fewer. In some embodiments, the sample can be asingle-cell sample. In some embodiments, the sample can comprise cellsisolated from a fine-needle aspirate.

Where the sample is a biological sample, in some aspects, the methods,systems and kits described herein can be used in personalized treatment.For example, a biological sample can be collected from an individualsubject who is in need of a treatment for a condition. Using themethods, systems and/or kits described herein, an expression profile oftarget molecules associated with the subject's condition can begenerated to identify one or more therapeutic targets for the subject,thereby identifying a treatment regimen for the subject. Accordingly,methods for identifying a treatment regimen for an individual subjectare also provided herein. In this aspect, the method comprises: (i)contacting a sample derived from a subject who is in need of a treatmentfor a condition, with a composition comprising a plurality of targetprobes that bind to target molecules associated with the condition; (ii)releasing the identification nucleotide sequences from the bound targetprobes; (iii) detecting signals from the released identificationnucleotide sequences, wherein the signals are distinguishable for theidentification nucleotide sequences, thereby identifying thecorresponding target-binding molecules and determining the presence ofone or more target molecules in the sample; and (iv) generating anexpression profile of the target molecules detected by the targetprobes, thereby selecting a treatment regimen for the individual subjectbased on the expression profile. The methods can be applied to anycondition described in the later section. In some embodiments, thecondition is cancer. In some embodiments, the signals from the releasedidentification nucleotide sequences are not detected by gelelectrophoresis-based methods.

In some aspects, the methods, systems and kits described herein can beused to assess how drug dosing corresponds to cellular pharmacodynamicsand thus used in monitoring response of a subject to a treatment forhis/her condition. For example, biological sample(s) can be collectedfrom the subject prior to and/or over the course of the treatment. Usingthe methods, systems and/or kits described herein, expression profilesof target molecules associated with the subject's condition beforeand/or over the course of the treatment can be generated for comparisonto determine any changes in expression levels of the target molecules inthe subject, thereby monitoring the treatment response in the subject.Accordingly, another aspect provided herein relates to a method ofmonitoring a treatment for a condition in a subject. The methodcomprises: (i) contacting a sample derived from a subject after atreatment for a condition, with a composition comprising a plurality oftarget probes that bind to target molecules associated with thecondition; (ii) releasing the identification nucleotide sequences fromthe bound target probes; (iii) detecting signals from the releasedidentification nucleotide sequences, wherein the signals aredistinguishable for the released identification nucleotide sequences,thereby identifying the corresponding target-binding molecules anddetermining the presence of one or more target molecules in the sample;(iv) generating an expression profile of the target molecules detectedby the target probes; (v) comparing the expression profile with anexpression profile generated from a sample derived from the same subjectprior to the treatment or after treatment at an earlier time point; and(vi) determining changes in expression levels of the target molecules,thereby monitoring the treatment for the condition in the subject. Insome embodiments, the signals from the released identificationnucleotide sequences are not detected by gel electrophoresis-basedmethods.

In some embodiments, the method can further comprise administering analternative treatment for the condition, when there are no substantialchanges in expression levels of the target molecules or the changes inexpression levels of the target molecules do not represent a reductionin symptoms associated with the condition.

In some embodiments, the method can further comprise continuing the sametreatment for the condition, when the changes in expression levels ofthe target molecules represent a reduction in symptoms associated withthe condition.

In some aspects, the methods, systems and kits described herein can beused in diagnosing a condition in a subject. For example, a biologicalsample can be collected from a subject who is at risk for a condition.Using the methods, systems and/or kits described herein, an expressionprofile of target molecules associated with the condition to bediagnosed can be generated for comparison with one or more referenceexpression profiles (e.g., corresponding to a normal healthy subjectand/or a subject having the condition to be diagnosed), therebydetermining whether the subject is at risk for the condition.Accordingly, provided here is also a method for diagnosing a conditionin a subject. The method comprises: (i) contacting a sample derived froma subject who is at risk for a condition, with a composition comprisinga plurality of target probes that bind to target molecules associatedwith the condition; (ii) releasing the identification nucleotidesequences from the bound target probes; (iii) detecting signals from thereleased identification nucleotide sequences, wherein the signals aredistinguishable for the identification nucleotide sequences, therebyidentifying the corresponding target-binding molecules and determiningthe presence of one or more target molecules in the sample; (iv)generating an expression profile of the target molecules detected by thetarget probes, (v) comparing the expression profile to at least onereference expression profile, thereby determining whether the subject isat risk for the condition. In some embodiments, the signals from thereleased identification nucleotide sequences are not detected by gelelectrophoresis-based methods.

In some embodiments, a reference expression profile is associated withthe condition. In some embodiments, a reference expression profile isassociated with a normal healthy subject.

In some embodiments, the methods described herein can be used todetermine intratumoral heterogeneity, which can be used as a biomarkerfor diagnosis and/or prognosis.

Conditions (e.g., Diseases or Disorders) Amenable to Diagnosis,Prognosis/Monitoring, and/or Treatment Using Methods, Systems, Kits, orVarious Aspects Described Herein

Different embodiments of the methods, systems and/or kits describedherein can be used for diagnosis and/or treatment of a disease ordisorder in a subject, e.g., a condition afflicting a certain tissue ina subject. For example, the disease or disorder in a subject can beassociated with breast, pancreas, blood, prostate, colon, lung, skin,brain, ovary, kidney, oral cavity, throat, cerebrospinal fluid, liver,or other tissues, and any combination thereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a breast disease or disorder. Exemplary breast disease ordisorder includes breast cancer.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a pancreatic disease or disorder. Nonlimiting examples ofpancreatic diseases or disorders include acute pancreatitis, chronicpancreatitis, hereditary pancreatitis, pancreatic cancer (e.g.,endocrine or exocrine tumors), etc., and any combinations thereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a blood disease or disorder. Examples of blood disease ordisorder include, but are not limited to, platelet disorders, vonWillebrand diseases, deep vein thrombosis, pulmonary embolism, sicklecell anemia, thalassemia, anemia, aplastic anemia, fanconi anemia,hemochromatosis, hemolytic anemia, hemophilia, idiopathicthrombocytopenic purpura, iron deficiency anemia, pernicious anemia,polycythemia vera, thrombocythemia and thrombocytosis, thrombocytopenia,and any combinations thereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a prostate disease or disorder. Non-limiting examples of aprostate disease or disorder can include prostatis, prostatichyperplasia, prostate cancer, and any combinations thereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a colon disease or disorder. Exemplary colon diseases ordisorders can include, but are not limited to, colorectal cancer,colonic polyps, ulcerative colitis, diverticulitis, and any combinationsthereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a lung disease or disorder. Examples of lung diseases ordisorders can include, but are not limited to, asthma, chronicobstructive pulmonary disease, infections, e.g., influenza, pneumoniaand tuberculosis, and lung cancer.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a skin disease or disorder, or a skin condition. An exemplaryskin disease or disorder can include skin cancer.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a brain or mental disease or disorder (or neural disease ordisorder). Examples of brain diseases or disorders (or neural disease ordisorder) can include, but are not limited to, brain infections (e.g.,meningitis, encephalitis, brain abscess), brain tumor, glioblastoma,stroke, ischemic stroke, multiple sclerosis (MS), vasculitis, andneurodegenerative disorders (e.g., Parkinson's disease, Huntington'sdisease, Pick's disease, amyotrophic lateral sclerosis (ALS), dementia,and Alzheimer's disease), Timothy syndrome, Rett symdrome, Fragile X,autism, schizophrenia, spinal muscular atrophy, frontotemporal dementia,any combinations thereof.

In some embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude a liver disease or disorder. Examples of liver diseases ordisorders can include, but are not limited to, hepatitis, cirrhosis,liver cancer, biliary cirrhosis, fatty liver, nonalcoholicsteatohepatitis (NASH), fibrosis, primary sclerosing cholangitis,Budd-Chiari syndrome, hemochromatosis, transthyretin-related hereditaryamyloidosis, Gilbert's syndrome, and any combinations thereof.

In other embodiments, the condition (e.g., disease or disorder) amenableto diagnosis and/or treatment using any aspects described herein caninclude cancer. A “cancer” or “tumor” as used herein refers to anuncontrolled growth of cells which interferes with the normalfunctioning of the bodily organs and systems. A subject that has acancer or a tumor is a subject having objectively measurable cancercells present in the subject's body. Included in this definition arebenign and malignant cancers, as well as dormant tumors ormicrometastases. Cancers which migrate from their original location andseed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organs. Hemopoieticcancers, such as leukemia, are able to out-compete the normalhemopoietic compartments in a subject, thereby leading to hemopoieticfailure (in the form of anemia, thrombocytopenia and neutropenia)ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

Metastases are most often detected through the sole or combined use ofmagnetic resonance imaging (MRI) scans, computed tomography (CT) scans,blood and platelet counts, liver function studies, chest X-rays and bonescans in addition to the monitoring of specific symptoms.

Examples of cancer include, but are not limited to carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer; cancer of the peritoneum; cervical cancer; choriocarcinoma;colon and rectum cancer; connective tissue cancer; cancer of thedigestive system; endometrial cancer; esophageal cancer; eye cancer;cancer of the head and neck; gastric cancer (including gastrointestinalcancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

In some embodiments, the methods and systems described herein can beused for determining in a subject a given stage of cancer, e.g., basedon the expression profiling generated using the methods describedherein. The stage of a cancer generally describes the extent the cancerhas progressed and/or spread. The stage usually takes into account thesize of a tumor, how deeply the tumor has penetrated, whether the tumorhas invaded adjacent organs, how many lymph nodes the tumor hasmetastasized to (if any), and whether the tumor has spread to distantorgans. Staging of cancer is generally used to assess prognosis ofcancer as a predictor of survival, and cancer treatment is primarilydetermined by staging.

Sample

In accordance with various embodiments described herein, a sample,including any fluid or specimen (processed or unprocessed) or otherbiological sample, can be subjected to the methods of various aspectsdescribed herein.

In some embodiments, the sample can include a biological fluid obtainedfrom a subject. Exemplary biological fluids obtained from a subject caninclude, but are not limited to, blood (including whole blood, plasma,cord blood and serum), lactation products (e.g., milk), amniotic fluids(e.g., a sample collected during amniocentesis), sputum, saliva, urine,peritoneal fluid, pleural fluid, semen, cerebrospinal fluid, bronchialaspirate, perspiration, mucus, liquefied feces or stool samples,synovial fluid, lymphatic fluid, tears, tracheal aspirate, and fractionsthereof. In some embodiments, a biological fluid can include ahomogenate of a tissue specimen (e.g., biopsy) from a subject. In oneembodiment, a test sample can comprises a suspension obtained fromhomogenization of a solid sample obtained from a solid organ or afragment thereof. In some embodiments, a sample can be obtained from amucosal swab. In some embodiments, a sample can be obtained from atissue biopsy (e.g. but not limited to skin biopsy). In someembodiments, a sample can be a fine needle aspirate.

In some embodiments, a sample can be obtained from a subject who has oris suspected of having a disease or disorder. In some embodiments, thesample can be obtained from a subject who has or is suspected of havingcancer, or who is suspected of having a risk of developing cancer.

In some embodiments, a sample can be obtained from a subject who isbeing treated for a disease or disorder. In other embodiments, thesample can be obtained from a subject whose previously-treated diseaseor disorder is in remission. In other embodiments, the test sample canbe obtained from a subject who has a recurrence of a previously-treateddisease or disorder. For example, in the case of cancer such as breastcancer, a test sample can be obtained from a subject who is undergoing acancer treatment, or whose cancer was treated and is in remission, orwho has cancer recurrence.

As used herein, a “subject” can mean a human or an animal. Examples ofsubjects include primates (e.g., humans, and monkeys). Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, and avian species,e.g., chicken, emu, ostrich. A patient or a subject includes any subsetof the foregoing, e.g., all of the above, or includes one or more groupsor species such as humans, primates or rodents. In certain embodimentsof the aspects described herein, the subject is a mammal, e.g., aprimate, e.g., a human. The terms, “patient” and “subject” are usedinterchangeably herein. A subject can be male or female. The term“patient” and “subject” does not denote a particular age. Thus, anymammalian subjects from adult to newborn subjects, as well as fetuses,are intended to be covered.

In one embodiment, the subject or patient is a mammal. The mammal can bea human, non-human primate, mouse, rat, dog, cat, horse, or cow, but arenot limited to these examples. In one embodiment, the subject is a humanbeing. In another embodiment, the subject can be a domesticated animaland/or pet.

In some embodiments, a sample that can be analyzed by the methods,systems and kits described herein can be obtained from an environmentalsource. Examples of an environmental source include, but are not limitedto, water, soil, food products, ponds, reservoir, and any combinationsthereof.

Linkers

As used herein, the term “linker” generally refers to a molecular entitythat can directly or indirectly connect at two parts of a composition.For example, in some embodiments with respect to a reporter probe, thelinker directly or indirectly connects a first target probe-specificregion to a detectable label described herein. In some embodiments withrespect to a capture probe, the linker directly or indirectly connects asecond target probe-specific region to an affinity tag described herein.In some embodiments with respect to a target probe, the linker directlyor indirectly connects an identification nucleotide sequence to atarget-binding molecule. In some embodiments with respect to a controlprobe, the linker directly or indirectly connects an identificationcontrol sequence to a control-binding molecule.

In some embodiments, a linker can comprise a peptide or nucleic acidlinker. The peptide or nucleic acid linker can be configured to have asequence comprising at least one of the amino acids selected from thegroup consisting of glycine (Gly), serine (Ser), asparagine (Asn),threonine (Thr), methionine (Met) or alanine (Ala), or at least one ofcodon sequences encoding the aforementioned amino acids (i.e., Gly, Ser,Asn, Thr, Met or Ala). Such amino acids and corresponding nucleic acidsequences are generally used to provide flexibility of a linker.However, in some embodiments, other uncharged polar amino acids (e.g.,Gln, Cys or Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro, Phe,and Trp), or nucleic acid sequences encoding the amino acids thereof canalso be included in a linker sequence. In alternative embodiments, polaramino acids or nucleic acid sequence thereof can be added to modulatethe flexibility of a linker. One of skill in the art can controlflexibility of a linker by varying the types and numbers of residues inthe linker. See, e.g., Perham, 30 Biochem. 8501 (1991); Wriggers et al.,80 Biopolymers 736 (2005).

In alternative embodiments, a linker can comprise a chemical linker ofany length. In some embodiments, chemical linkers can comprise a directbond or an atom such as oxygen or sulfur, a unit such as NH, C(O),C(O)NH, SO, SO₂, SO₂NH, or a chain of atoms, such as substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, C(O)N(R¹)₂, C(O), cleavable linker,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R¹ ishydrogen, acyl, aliphatic or substituted aliphatic. In some embodiments,the chemical linker can be a polymer chain (branched or linear).

In some embodiments, the chemical linker can comprise a stable or labile(e.g., cleavable) bond or conjugation agent. Exemplary conjugationsinclude, but are not limited to, covalent bond, amide bond, additions tocarbon-carbon multiple bonds, azide alkyne Huisgen cycloaddition,Diels-Alder reaction, disulfide linkage, ester bond, Michael additions,silane bond, urethane, nucleophilic ring opening reactions: epoxides,non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolarcycloaddition, temperature sensitive, radiation (IR, near-IR, UV)sensitive bond or conjugation agent, pH-sensitive bond or conjugationagent, non-covalent bonds (e.g., ionic charge complex formation,hydrogen bonding, pi-pi interactions, cyclodextrin/adamantly host guestinteraction) and the like.

As used herein, the term “conjugation agent” means an organic moietythat connects two parts of a compound. Without limitations, anyconjugation chemistry known in the art for conjugating two molecules ordifferent parts of a composition together can be used for coupling twoparts of a compound. Exemplary coupling molecules and/or functionalgroups for coupling two parts of a compound include, but are not limitedto, a polyethylene glycol (PEG, NH2-PEGX—COOH which can have a PEGspacer arm of various lengths X, where 1<X<100, e.g., PEG-2K, PEG-5K,PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K, and the like), maleimideconjugation agent, PASylation, HESylation, Bis(sulfosuccinimidyl)suberate conjugation agent, DNA conjugation agent, peptide conjugationagent, silane conjugation agent, hydrolyzable conjugation agent, and anycombinations thereof.

In some embodiments, the linker includes a coupling molecule pair. Theterms “coupling molecule pair” and “coupling pair” as usedinterchangeably herein refer to the first and second molecules thatspecifically bind to each other. One member of the coupling pair isconjugated to a first entity while the second member is conjugated to asecond entity, which is desired to be connected to the first entity. Byway of example only, the first entity can be a detectable label of areporter probe described herein, and the second entity can be a firsttarget probe-specific region of the reporter probe. Thus, the detectablelabel can be coupled to the first target probe-specific region via acoupling molecule pair. As another example, a solid substrate surfacecan comprise a first member of the coupling pair, while an affinity tagof a capture probe described here can comprise a second member of thecoupling pair. As used herein, the phrase “first and second moleculesthat specifically bind to each other” refers to binding of the firstmember of the coupling pair to the second member of the coupling pairwith greater affinity and specificity than to other molecules.

Exemplary coupling molecule pairs include, without limitations, anyhaptenic or antigenic compound in combination with a correspondingantibody or binding portion or fragment thereof (e.g., digoxigenin andanti-digoxigenin; mouse immunoglobulin and goat antimouseimmunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin,biotin-streptavidin), hormone (e.g., thyroxine and cortisol-hormonebinding protein), receptor-receptor agonist, receptor-receptorantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme inhibitor, and complementary oligonucleotide pairs capableof forming nucleic acid duplexes). The coupling molecule pair can alsoinclude a first molecule that is negatively charged and a secondmolecule that is positively charged.

One example of using coupling pair conjugation is the biotin-avidin orbiotin-streptavidin conjugation. In this approach, a first entity isbiotinylated (i.e., the first entity comprise a biotin molecule) and asecond entity desired to be connected to the first entity can comprisean avidin or streptavidin. Many commercial kits are also available forbiotinylating molecules, such as proteins. For example, anaminooxy-biotin (AOB) can be used to covalently attach biotin to amolecule with an aldehyde or ketone group.

Still another example of using coupling pair conjugation isdouble-stranded nucleic acid conjugation. In this approach, a firstentity can comprise a first strand of the double-stranded nucleic acidand a second entity desired to be connected to the first entity cancomprise a second strand of the double-stranded nucleic acid. Nucleicacids can include, without limitation, defined sequence segments andsequences comprising nucleotides, ribonucleotides, deoxyribonucleotides,nucleotide analogs, modified nucleotides and nucleotides comprisingbackbone modifications, branchpoints and nonnucleotide residues, groupsor bridges.

In some embodiments, a linker can be a physical substrate, e.g.,microparticles or magnetic particles.

The linkers can be of any shape. In some embodiments, the linkers can belinear. In some embodiments, the linkers can be folded. In someembodiments, the linkers can be branched. In other embodiments, thelinker adopts the shape of the physical substrate.

In some embodiments, the linker can comprise a cleavable linkerdescribed herein.

Embodiments of Various Aspects Described Herein can be Defined in any ofthe Following Numbered Paragraphs:

-   1. A method for detecting a plurality of target molecules in a    sample comprising:    -   a. contacting a sample with a composition comprising a plurality        of target probes, wherein each target probe in the plurality        comprises:        -   i. a target-binding molecule that specifically binds to a            distinct target molecule in the sample;        -   ii. an identification nucleotide sequence that identifies            the target-binding molecule; and        -   iii. a cleavable linker between the target-binding molecule            and the identification nucleotide sequence;    -   b. separating unbound target probes from a plurality of        complexes in the sample, each complex having a target molecule        and a single target probe bound thereto, wherein the complex        does not have a second target probe binding to a different        region of the target molecule;    -   c. releasing the identification nucleotide sequences from the        plurality of complexes;    -   d. detecting signals from the released identification nucleotide        sequences based on a non-gel electrophoresis method, wherein the        signals are distinguishable for the identification nucleotide        sequences, thereby identifying the corresponding target-binding        molecules and detecting a plurality of different target        molecules in the sample.-   2. The method of paragraph 1, wherein the non-gel electrophoresis    method comprises sequencing, quantitative polymerase chain reaction    (PCR), multiplexed (PCR), mass cytometry, fluorophore-inactivated    multiplexed immunofluorescence, hybridization-based methods,    fluorescence hybridization-based methods, or any combinations    thereof.-   3. The method of paragraph 1 or 2, further comprising, prior to the    detecting step (d), coupling the released identification nucleotide    sequences from the releasing step (c) to a detection composition    comprising a plurality of reporter probes, wherein each reporter    probe in the plurality comprises: a first target probe-specific    region that is capable of binding a first portion of the    identification nucleotide sequence; and a detectable label that    identifies the reporter probe.-   4. The method of paragraph 3, wherein the detecting comprises    detecting signals from the respective detectable labels of the    reporter probes that are coupled to the released identification    nucleotide sequences, wherein the signals are distinguishable for    the respective reporter probes and bound the identification    nucleotide sequences, thereby identifying the corresponding    target-binding molecules and detecting a plurality of target    molecules in the sample.-   5. The method of paragraph 3 or 4, wherein the detection composition    further comprises a plurality of capture probes, wherein each    capture probe comprises (i) a second target probe-specific region    that is capable of binding a second portion of the identification    nucleotide sequence; and (ii) an affinity tag.-   6. The method of paragraph 5, wherein the affinity tag of the    capture probe permits immobilization of the released identification    nucleotide sequences onto a solid substrate, upon coupling to the    detection composition.-   7. The method of any of paragraphs 3-6, wherein the detectable label    of the reporter probes comprises one or more labeling molecules that    create a unique signal for each reporter probe.-   8. The method of paragraph 7, wherein the unique signal is an    optical signal.-   9. The method of paragraph 8, wherein the optical signal comprises a    sequence of light-emitting signals.-   10. The method of any of paragraphs 7-9, wherein the one or more    labeling molecules are selected from the group consisting of a    fluorochrome moiety, a fluorescent moiety, a dye moiety, a    chemiluminescent moiety, and any combinations thereof.-   11. The method of any of paragraphs 1-10, wherein the detecting    step (d) comprises no amplification of the released identification    nucleotide sequences.-   12. The method of any of paragraphs 3-11, wherein the detecting    step (d) comprises no amplification of the first target    probe-specific region, or the second target probe-specific region.-   13. The method of any of paragraphs 1-12, wherein the identification    nucleotide sequences are selected such that they do not cross-react    with a human genome.-   14. The method of paragraph 13, wherein the identification    nucleotide sequences are derived from a potato genome.-   15. The method of any of paragraphs 1-14, wherein the identification    nucleotide sequences have a length of about 30-100 nucleotides.-   16. The method of any of paragraphs 1-15, wherein the identification    nucleotide sequences have a length of about 70 nucleotides.-   17. The method of paragraph 16, wherein the identification    nucleotide sequences have a sequence selected from Table 2 (SEQ ID    NO: 1 to SEQ ID NO: 110).-   18. The method of any of paragraphs 1-17, wherein the cleavable    linker is a cleavable, non-hybridizable linker.-   19. The method of paragraph 18, wherein the cleavable,    non-hybridizable linker is sensitive to an enzyme, pH, temperature,    light, shear stress, sonication, a chemical agent (e.g.,    dithiothreitol), or any combination thereof.-   20. The method of paragraph 18 or 19, wherein the cleavable,    non-hybridizable linkers are selected from the group consisting of    hydrolyzable linkers, redox cleavable linkers, phosphate-based    cleavable linkers, acid cleavable linkers, ester-based cleavable    linkers, peptide-based cleavable linkers, photocleavable linkers,    and any combinations thereof.-   21. The method of any of paragraphs 18-20, wherein the cleavable,    non-hybridizable linker comprises a disulfide bond, a    tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl    group, a nitoindoline group, a bromo hydroxycoumarin group, a bromo    hydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin    group, or any combinations thereof.-   22. The method of any of paragraphs 18-21, wherein the cleavable,    non-hybridizable linker comprises a photocleavable linker.-   23. The method of paragraph 22, wherein the photocleavable linker is    selected from the group consisting of molecules (i)-(xiv) and any    combinations thereof, wherein the chemical structures of the    molecules (i)-(xiv) are shown as follows:

wherein each of the black dots in each molecule represents a connectingor coupling point that connects, directly or indirectly, to thetarget-binding molecule or the identification nucleotide sequence.

-   24. The method of paragraph 22, wherein the photocleavable linker    comprises the molecule (xiv).-   25. The method of any of paragraphs 22-24, wherein the releasing of    the identification nucleotide sequences from the bound target probes    comprises exposing the bound target probes to ultraviolet light.-   26. The method of any of paragraphs 1-25, wherein the sample    comprises less than 500 cells.-   27. The method of any of paragraphs 1-26, wherein the sample is a    single-cell sample.-   28. The method of any of paragraphs 1-27, wherein the sample    comprises cells isolated from a fine-needle aspirate.-   29. The method of any of paragraphs 1-28, further comprising, prior    to the contacting, separating target cells from interfering cells in    the sample.-   30. The method of paragraph 29, wherein the separating comprises    labeling the interfering cells or target cells with magnetic    particles and separating them from the sample by magnetic    separation.-   31. The method of paragraph 30, wherein the magnetic separation is    performed in a microfluidic device.-   32. The method of any of paragraphs 29-31, wherein the target cells    comprise rare cells.-   33. The method of paragraph 32, wherein the rare cells are selected    from the group consisting of circulating tumor cells, fetal cells,    stem cells, immune cells, clonal cells, and any combination thereof.-   34. The method of any of paragraphs 29-33, wherein the target cells    comprise tumor cells from a liquid biopsy (e.g., peritoneal,    pleural, cerebrospinal fluid, and/or blood), a mucosal swap, a skin    biopsy, a stool sample, or any combinations thereof.-   35. The method of any of paragraphs 1-34, wherein the target    molecules comprise proteins, peptides, metabolites, lipids,    carbohydrates, toxins, growth factors, hormones, cytokines, cells,    and any combinations thereof.-   36. The method of any of paragraphs 1-35, further comprising    permeabilizing the target cells in the sample.-   37. The method of any of paragraphs 1-36, wherein the composition    further comprises a plurality of control probes, wherein each    control probe in the plurality comprises:    -   i. a control-binding molecule that specifically binds to one        control molecule in the sample;    -   ii. an identification control sequence that identifies the        control-binding molecule; and    -   iii. a cleavable linker between the control-binding molecule and        the identification control sequence.-   38. The method of paragraph 37, wherein the control-binding molecule    binds to a control protein.-   39. The method of paragraph 38, wherein the control protein is    selected from the group consisting of housekeeping proteins, control    IgG isotypes, mutant non-functional or non-binding proteins, and any    combinations thereof.-   40. The method of any of paragraphs 1-39, further comprising    thresholding the signals.-   41. The method of paragraph 40, wherein the signals are thresholded    on the basis of nonspecific binding.-   42. The method of paragraph 41, wherein the threshold is greater    than that of the signals from the non-specific binding.-   43. The method of any of paragraphs 40-42, wherein the threshold is    determined by using standard deviation and measurement error from at    least one control protein.-   44. The method of any of paragraphs 1-43, further comprising    quantifying the signals by normalizing the signals associated with    the target probes by the signals associated with the control probes.-   45. The method of any of paragraphs 1-44, further comprising    extracting a nucleic acid molecule from the same sample for nucleic    acid analysis.-   46. The method of paragraph 45, further comprising subjecting the    nucleic acid molecule to a nucleic acid analysis.-   47. The method of paragraph 46, wherein the nucleic acid analysis    comprises sequencing, quantitative polymerase chain reaction (PCR),    multiplexed PCR, DNA sequencing, RNA sequencing, de novo sequencing,    next-generation sequencing such as massively parallel signature    sequencing (MPSS), polony sequencing, pyrosequencing, Illumina    (Solexa) sequencing, SOLiD sequencing, ion semiconductor sequencing,    DNA nanoball sequencing, Heliscope single molecule sequencing,    single molecule real time (SMRT) sequencing, nanopore DNA    sequencing, sequencing by hybridization, sequencing with mass    spectrometry, microfluidic Sanger sequencing, microscopy-based    sequencing techniques, RNA polymerase (RNAP) sequencing, or any    combinations thereof.-   48. The method of any of paragraphs 45-47, wherein the target    molecules to be detected in the sample comprise proteins, thereby    detecting proteins and nucleic acid molecules from the same sample.-   49. A kit for multiplexed detection of a plurality of different    target molecules from a sample comprising:    -   a. a plurality of target probes, wherein each target probe in        the plurality comprises:        -   i. a target-binding molecule that specifically binds to a            distinct target molecule in the sample;        -   ii. an identification nucleotide sequence that identifies            the target-binding molecule; and        -   iii. a cleavable, non-hybridizable linker between the            target-binding molecule and the identification nucleotide            sequence;    -   b. a plurality of reporter probes, wherein each reporter probe        comprises:        -   i. a first target probe-specific region that is capable of            binding a first portion of the identification nucleotide            sequence; and        -   ii. a detectable label that identifies the reporter probe.-   50. The kit of paragraph 49, further comprising a plurality of    capture probes, wherein each capture probe comprises (i) a second    target probe-specific region that is capable of binding a second    portion of the identification nucleotide sequence; and (ii) an    affinity tag.-   51. The kit of paragraph 49 or 50, wherein the detectable label of    the reporter probes comprises one or more labeling molecules that    create a unique signal for each reporter probe.-   52. The kit of paragraph 51, wherein the unique signal is an optical    signal.-   53. The kit of paragraph 52, wherein the optical signal comprises a    sequence of light-emitting signals.-   54. The kit of any of paragraphs 51-53, wherein the one or more    labeling molecules are selected from the group consisting of a    fluorochrome moiety, a fluorescent moiety, a dye moiety, a    chemiluminescent moiety, and any combinations thereof.-   55. The kit of any of paragraphs 49-54, wherein the target-binding    molecule comprises proteins, peptides, metabolites, lipids,    carbohydrates, toxins, growth factors, hormones, cytokines, cells,    and any combination thereof.-   56. The kit of any of paragraphs 49-55, wherein the cleavable,    non-hybridizable linker is sensitive to an enzyme, pH, temperature,    light, shear stress, sonication, a chemical agent (e.g.,    dithiothreitol), or any combination thereof.-   57. The kit of any of paragraphs 49-56, wherein the cleavable,    non-hybridizable linkers are selected from the group consisting of    hydrolyzable linkers, redox cleavable linkers, phosphate-based    cleavable linkers, acid cleavable linkers, ester-based cleavable    linkers, peptide-based cleavable linkers, photocleavable linkers,    and any combinations thereof.-   58. The kit of any of paragraphs 49-57, wherein the cleavable,    non-hybridizable linker comprises a disulfide bond, a    tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl    group, a nitoindoline group, a bromo hydroxycoumarin group, a bromo    hydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin    group, or any combinations thereof.-   59. The kit of any of paragraphs 49-58, wherein the cleavable,    non-hybridizable linker comprises a photocleavable linker.-   60. The kit of paragraph 59, wherein the photocleavable linker is    selected from the group consisting of molecules (i)-(xiv) and any    combinations thereof, wherein the chemical structures of the    molecules (i)-(xiv) are shown as follows:

wherein each of the black dots in each molecule represents a connectingor coupling point that connects, directly or indirectly, to thetarget-binding molecule or the identification nucleotide sequence.

-   61. The kit of paragraph 59, wherein the photocleavable linker    comprises the molecule (xiv).-   62. The kit of any of paragraphs 49-61, further comprising a    plurality of control probes, wherein each control probe in the    plurality comprises:    -   i. a control-binding molecule that specifically binds to one        control molecule in the sample;    -   ii. an identification control sequence that identifies the        control-binding molecule; and    -   iii. a cleavable linker between the control-binding molecule and        the identification control sequence.-   63. The kit of paragraph 62, wherein the control-binding molecule    binds to a control protein.-   64. The kit of paragraph 63, wherein the control protein is selected    from the group consisting of housekeeping proteins, control IgG    isotypes, mutant non-functional or non-binding proteins, and any    combinations thereof.-   65. The kit of any of paragraphs 49-64, further comprising a reagent    selected from the group consisting of a hybridization reagent, a    purification reagent, an immobilization reagent, an imaging agent, a    cell permeabilization agent, a blocking agent, a cleaving agent for    the cleavable linker, and any combinations thereof.-   66. The kit of any of paragraphs 49-65, further comprising a device,    wherein the device comprises a surface for immobilization of the    capture probes upon coupling to the identification nucleotide    sequences.    Some Selected Definitions

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” with respect to numerical values means within5%.

As used herein, the term “comprising” or “comprise(s)” is used inreference to compositions, methods, and respective component(s) thereof,that are essential to the invention, yet open to the inclusion ofunspecified elements, whether essential or not.

As used herein, the term “consisting essentially of” or “consist(s)essentially of” refers to those elements required for a givenembodiment. The term permits the presence of additional elements that donot materially affect the basic and novel or functionalcharacteristic(s) of that embodiment of the invention.

As used herein, the term “consisting of” or “consist(s) of” refers tocompositions, methods, and respective components thereof as describedherein, which are exclusive of any element not recited in thatdescription of the embodiment.

The term “multiplexed detection” refers to detection of a plurality oftarget molecules from a single sample in a single assay. In someembodiments, multiplexed detection refers to simultaneous measurementsof at least 2, at least 3, at least 4, at least 5, at least 10, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100, at least 150, at least 200, atleast 250, at least 300, at least 350, at least 400, at least 450, atleast 500 or more different target molecules from a single sample.

As used herein, the term “fixed cell or tissue sample” refers to asample obtained from a cell or tissue which has been previously fixed ina cell- or tissue-fixing solution and optionally afterwards embedded ina solid substrate. Various cell- or tissue-fixing solutions are known inthe art, including, but not limited to aldehydes (e.g., but not limitedto formaldehyde, formalin), alcohols (e.g., but not limited to, ethanol,methanol, and/or acetone), oxidizing agents (e.g., but not limited to,osmium tetroxide, potassium dichromate, chromic acid, and/or potassiumpermanganate), picrates, mercurial (e.g., but not limited to, B-5 and/orZenker's fixative), Hepes-glutamic acid buffer-mediated Organic solventProtection Effect (HOPE) fixative. In some embodiments, a fixed cell ortissue sample also encompasses a frozen cell or tissue sample.

As used herein, the term “alien or foreign DNA barcode” refers to a DNAsequence used as a barcode or tag for identification of a targetmolecule in a sample of an organism, wherein the DNA sequence is analien or foreign sequence relative to the genomes of the organism fromwhich the sample is derived or obtained. As used herein, the term “alienor foreign” refers to a nucleotide sequence that shows no or littlehomology against an organism (from which a sample is derived orobtained) and/or other major organisms, e.g., in the NCBI ReferenceSequence (RefSeq) database. In some embodiments, a nucleotide sequenceis alien or foreign when it shares a homology (sequence identity) withthat of the organism by no more than 50% or less, including, e.g., nomore than 40%, no more than 30%, no more than 20%, no more than 10% orless. In some embodiments, the identification nucleotide sequencesdescribed herein are alien or foreign DNA barcodes.

The term “antibody” as used herein refers to a full length antibody orimmunoglobulin, IgG, IgM, IgA, IgD or IgE molecules, or a proteinportion thereof that comprises only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind a target, such as an epitope orantigen. Examples of portions of antibodies or epitope-binding proteinsencompassed by the present definition include: (i) the Fab fragment,having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is aFab fragment having one or more cysteine residues at the C-terminus ofthe CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv)the Fd′ fragment having VH and CH1 domains and one or more cysteineresidues at the C terminus of the CH1 domain; (v) the Fv fragment havingthe VL and VH domains of a single arm of an antibody; (vi) the dAbfragment (Ward et al., 341 Nature 544 (1989)) which consists of a VHdomain or a VL domain that binds antigen; (vii) isolated CDR regions orisolated CDR regions presented in a functional framework; (viii) F(ab′)2fragments which are bivalent fragments including two Fab′ fragmentslinked by a disulphide bridge at the hinge region; (ix) single chainantibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242Science 423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x)“diabodies” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) inthe same polypeptide chain (see, e.g., EP 404,097; WO 93/11161;Hollinger et al., 90 PNAS 6444 (1993)); (xi) “linear antibodies”comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al., 8 Protein Eng. 1057 (1995); and U.S.Pat. No. 5,641,870).

“Antibodies” include antigen-binding portions of antibodies such asepitope- or antigen-binding peptides, paratopes, functional CDRs;recombinant antibodies; chimeric antibodies; tribodies; midibodies; orantigen-binding derivatives, analogs, variants, portions, or fragmentsthereof.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a target molecule, orto a molecule in a signaling pathway that modulates the expressionand/or activity of a target molecule. The generation and therapeutic useof aptamers are well established in the art. See, e.g., U.S. Pat. No.5,475,096.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

EXAMPLES Example 1. Optimization for Methods for Multiplex Detection ofTarget Molecules from a Sample

Evaluation of different cleavable linkers: In various embodimentsdescribed herein, target-binding molecules can be conjugated toidentification nucleotide sequences via any cleavable linker(s) known inthe art. In this Example, three alternative methods of conjugatingtarget molecules (e.g., antibodies) to identification nucleotidesequences (e.g., DNA) via a cleavable linker were evaluated usingexemplary procedures detailed below.

In the first method, target-binding molecules (e.g., antibodies) weremodified with (E)-Cyclooct-4-enyl 2,5-dioxopyrro-lidin-1-yl carbonate(trans-cyclooctene N-hydroxy-succinimidyl ester; TCO-NHS) andsynthesized as previously reported in Ref. 26. If present, sodium azidewas removed using a 2 ml Zeba desalting column (7 K MWCO). The reactionwas performed using 1000 molar equivalents of TCO-NHS in PBS containing10% (v/v) DMF and 10 mM sodium bicarbonate for 4 h at RT. At the sametime, a photocleavable Tz-NHS was reacted with an amine group on the 5′end of the 70mer DNA strand (15 molar excess) for 4 h at RT. After thereactions concluded, the target-binding molecule-TCO (e.g., Ab-TCO)conjugate was purified using a Zeba column (7000 MWCO), and the DNA-Tzconjugate was purified using a 3 K MWCO Amicon filter followed by threewashes with PBS. Next, the target-binding molecule-TCO (e.g., Ab-TCO)and Tz-DNA were combined via click chemistry (26) for two hours at RT.The final target probe (e.g., antibody-DNA conjugate) was purified bysize separation using Amicon 100 K MWCO filters followed by washes withPBS.

In the second method, the photocleavable bifunctional linker (FIG. 2)reacted (10 molar excess) with the amine group on the 5′ end of the70mer single-stranded DNA (IDT) for 4 h at RT. Three hours after the DNAreaction began, the target-binding molecules (e.g., antibody) wasreacted with 2-iminothiolane (Traut's reagent, 10 molar excess, ThermoScientific) to convert amine groups to sulfydryl (—SH) groups in PBSwith 2 mM EDTA for 1 h at RT. When the reactions concluded, thethiolated target-binding molecules (e.g., antibody) was separated fromexcess Traut's Reagent using a Zeba desalting column (7000 MWCO) thathad been equilibrated with PBS containing 2 mM EDTA. The excessphotocleavable (PC) bifunctional linker was purified from the DNA with a3 K MWCO Amcion filter. Then the target-binding molecule-SH (e.g.,antibody-SH) and the DNA-PC-linker (˜15 molar excess) were reactedovernight at 4° C. The final target probe (e.g., antibody-DNA conjugate)was purified by size separation using Amicon 100 K MWCO filters followedby washes with PBS.

In the third method, an amine to sulfhydryl linker, sulfosuccinimidyl6-[3′(2-pyridyldithio)-propionamido] hexanoate (sulfo-LC-SPDP, ThermoScientific), was reacted with a target-binding molecule (e.g., antibody)in PBS-EDTA at 50 molar excess and aged for 1 h at RT. At the end of thereaction, excess sulfo-LC-SPDP was removed using a Zeba desalting column(7000 MWCO). The thiolyated DNA was reduced with DTT and purified via aNAP-5 column, as previously described in the DNA-antibody conjugation inthe “Exemplary materials and methods” section below. Once excesssulfo-LC-SPDP was purified using a Zeba column, the target-bindingmolecule (e.g., antibody) was reacted with the reduced thiolyated DNA(˜15 molar excess) overnight at 4° C. The final target probe (e.g.,antibody-DNA conjugate) was purified by size separation using Amicon 100K MWCO filters followed by washes with PBS.

The three UV-cleavable target-binding molecule-DNA (e.g., Ab-DNA) linkermethods were compared by first labeling A431 cells with EGFR and EPCAMDNA conjugates and then determining which method resulted in the highestsignal to noise ratio (SNR), e.g., via Nanostring. The conjugation oftarget molecules (e.g., antibodies) with the bifunctional photocleavablelinker described in FIG. 2A gave the highest SNR. This target probe(e.g., antibody-conjugate) was then compared to the target probe (e.g.,antibody-DNA conjugate containing the DTT cleavable disulfide bond.SKOV3 cells (5×10⁵ cells) were labeled with Herceptin-DNA conjugates (1μg). After 30 minutes the cells were spun down at 400×g for 3 minutes,and the excess Herceptin was removed with two SB+washes. TheHerceptin-DNA conjugate with the disulfide linker was then cleaved byadding DTT (50 mM) for 15 minutes at 37° C. At the same time, theHerceptin-DNA conjugate with the photocleavable linker was exposed to UVlight (wavelength) for 15 minutes. After the 15-minute cleavage step,the cells were spun down at 400×g for 5 minutes, and the supernatant wasremoved. The DNA in the supernatant was measured using thesingle-stranded Qubit assay to determine the amount of DNA cleaved fromthe antibody. The UV photocleavable linker had 2.4-fold more DNA thanthe disulfide linker.

Optimization of lysis conditions: Four different lysis conditions wereevaluated to determine which was the most efficient (FIG. 3): (A)Proteinase K with buffer PKD (Qiagen) and UV; (B) Proteinase K withbuffer ATL (Qiagen) and UV; (C) ATL buffer with UV; and (D) UV. Based onthe tested methods, method (B) showed a 20% increase in signal overmethods (A) and (C).

Example 2. Development and Validation of Methods for Multiplex Detectionof Target Molecules from a Sample

In this Example, an antibody barcoding with photocleavable DNA (ABCD)platform was designed to perform multiplexed protein measurements andsystem-wide profiling on small amounts of clinical sample material(e.g., ˜100 cells). The method was designed to preserve genetic materialand to enable specific isolation of rare single cells. This approachinterrogates single cells by tagging antibodies of interest with short(˜70-mer) DNA “barcodes”—with each antibody having a uniquesequence-using a stable photocleavable linker. Photocleavable linkersknown in the art (e.g., Ref. 9) can be used herein. After antibodybinding to the cells, the photocleavable linker releases the unique DNAbarcode, which can then be detected by various means. In someembodiments, different DNA barcodes can be identified based on sizeusing gel electrophoresis. However, this method had limited multiplexing(8 to 12 markers) and was only semiquantitative (9). Other quantitativemethods, such as sequencing and quantitative polymerase chain reaction(qPCR), are reliable and can be used to detect the released DNAbarcodes, but may introduce bias during amplification steps, requireprolonged processing time, or are not cost-effective. Multiplexed qPCRonly measures a maximum of five markers at a time. Thus, a fluorescencehybridization technology, which have been traditionally used formultiplexed quantitation (16,384 barcodes) of femtomolar amounts of DNAand RNA (10, 11), was selected to detect the released DNA barcodes.While the fluorescence hybridization technology has been used toquantify DNA and RNA, it had not been previously extended to measureproteins within cells or clinical samples. This Example and subsequentExamples show application and validation of the ABCD platform in celllines and human clinical specimens, as well as evaluating drug treatmentresponse and inter- and intrapatient heterogeneity in lung cancer.

Cells were first harvested from fine-needle aspirates (FNAs) from agiven patient (FIG. 1A). To better isolate cancer cells from theirheterogeneous cellular milieu, aspirates were labeled with antibodiesdirected against established markers (for example, CD45 to depletetumor-infiltrating leukocytes from the sample). The antibody was taggedwith magnetic nanoparticles and passed through a microfluidic devicecontaining a self-assembled magnetic layer to deplete tagged cells [12].The purified cancer cell population was retrieved from the device andstained with a mixture comprising a plurality of one or more embodimentsof target probes as described herein. In this Example, the purifiedcancer cell population were stained with a mixture of target probes eachcontaining an antibody and a unique barcode attached via aphotocleavable linker (referred to as “antibody conjugate” or“antibody-DNA conjugate” herein) (FIG. 1B and FIG. 2). Exampleantibodies for use in the antibody conjugates are listed in Table 1below. In this Example, more than 90 antibodies in the cocktail werechosen and used to demonstrate that bulk labeling yielded similarresults to single antibody labeling. The 90 antibody-DNA conjugates werespecially designed to tag an alien DNA sequence that would notcross-react with the human genome. Target markers were selected to coverhallmark pathways in cancer (e.g., apoptosis, epigenetic, and DNAdamage), cancer diagnostic markers known in the art, e.g., thosecommonly used in the clinic, and housekeeping and control proteins.Before labeling, antibody-DNA conjugates were isolated viaimmunoglobulin G (IgG)-specific pull-down and pooled together into acocktail. After cell blocking, permeabilization and labeling, andwashing, the DNA was released from the cells of interest with bothproteolytic cleavage and photocleavage to increase yield and, byextension, sensitivity (FIG. 1C).

TABLE 1 List of example antibodies. Antibody Species Catalog VendorGAPDH (14C10) Rabbit 2118BF Cell Signaling β-Tubulin (9F3) Rabbit 2128BFCell Signaling Ku80 (C48E7) Rabbit 2180BF Cell Signaling Phospho-Chk2(Thr68) (C13C1) Rabbit 2197BF Cell Signaling S6 ribosomal protein (54D2)Mouse 2317BF Cell Signaling Phospho-Chk1 (Ser345) (133D3) Rabbit 2348BFCell Signaling VE-cadherin (D87F2) Rabbit 2500BF Cell Signaling p53(7F5) Rabbit 2527BF Cell Signaling Phospho-53BP1 (Ser1778) Rabbit 2675BFCell Signaling Phospho-(Ser/Thr) ATM/ATR Substrate Rabbit 2851BF CellSignaling Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit 2855BF Cell SignalingBim (C34C5) Rabbit 2933BF Cell Signaling Cyclin D3 (DCS22 Mouse 2936BFCell Signaling Cyclin D1 (92G2) Rabbit 2978BF Cell Signaling mTOR (7C10)Rabbit 2983BF Cell Signaling Phospho-cyclin D1 (Thr286) (D29B3) Rabbit3300BF Cell Signaling Phospho-histone H3 (Ser10) (D2C8) Rabbit 3377BFCell Signaling ALK (D5F3) Rabbit 3633BF Cell Signaling Phospho-EGFReceptor (Tyr1068) (D7A5) Rabbit 3777BF Cell Signaling Phospho-Akt(Ser473) (D9E) Rabbit 4060BF Cell Signaling CDCP1 Antibody Rabbit 4115BFCell Signaling Cyclin E1 (HE12) Mouse 4129BF Cell SignalingPhospho-cyclin E1 (Thr62) Rabbit 4136BF Cell Signaling Phospho-p44/42MAPK (Erk1/2) (Thr202/ Rabbit 4370BF Cell Signaling Tyr204) (D13.14.4E)Keratin 7 (D1E4) Rabbit 4465BF Cell Signaling Histone H3 (D1H2) Rabbit4499BF Cell Signaling Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) Rabbit4511BF Cell Signaling Phospho-SEK1/MKK4 (Ser257) (C36C11) Rabbit 4514BFCell Signaling Pan-keratin (C11 Mouse 4545BF Cell Signaling Keratin 8/18(C51) Mouse 4546BF Cell Signaling Keratin 18 (DC10) Mouse 4548BF CellSignaling Akt (pan) (C67E7) Rabbit 4691BF Cell Signaling p44/42 MAPK(Erk1/2) (137F5 Rabbit 4695BF Cell Signaling COX IV (3E11 Rabbit 4850BFCell Signaling Phospho-S6 ribosomal protein (Ser235/236) Rabbit 4858BFCell Signaling 53BP1 Rabbit 4937BF Cell Signaling β-Actin (13E5) Rabbit4970BF Cell Signaling Akt2 (L79B2) Mouse 5239BF Cell SignalingPhospho-mTOR (Ser2448) (D9C2) Rabbit 5536BF Cell Signaling Cleaved PARP(Asp214) (D64E10) Rabbit 5625BF Cell Signaling Vimentin (D21H3) Rabbit5741BF Cell Signaling Cleaved caspase-9 (Asp330) (D2D4) Rabbit 7237BFCell Signaling Met (D1C2) Rabbit 8198BF Cell Signaling FGF receptor 4(D3B12) Rabbit 8562BF Cell Signaling Axl (C89E7) Rabbit 8661BF CellSignaling p38 MAPK (D13E1) Rabbit 8690BF Cell Signaling BRCA1 (D54A8)Rabbit 9025BF Cell Signaling Phospho-Stat3 (Tyr705) (D3A7) Rabbit 9145BFCell Signaling Cleaved caspase-7 (Asp198) Rabbit 9491BF Cell SignalingCleaved caspase-8 (Asp391) (18C8) Rabbit 9496BF Cell Signaling Cleavedcaspase-9 (Asp315) Rabbit 9505BF Cell Signaling PARP (46D11) Rabbit9532BF Cell Signaling 4E-BP1 (53H11) Rabbit 9644BF Cell SignalingCleaved caspase-3 (Asp175) Rabbit 9661BF Cell Signaling Phospho-histoneH2A.X (Ser139) (20E3) Rabbit 9718BF Cell Signaling FGF receptor 1 (D8E4)Rabbit 9740BF Cell Signaling Caspase-8 (1C12) Mouse 9746BF CellSignaling Caspase-9 Rabbit 9502 BF Cell Signaling Phospho-β-Catenin(Ser675) (D2F1) Rabbit 4176BF Cell Signaling Phospho-GSK-3β (Ser9)(D85E12) Rabbit 5558BF Cell Signaling Dimethyl-Histone H3 (Lys9) (D85B4)Rabbit 4658BF Cell Signaling Dimethyl-Histone H3 (Lys4) (C64G9) Rabbit9725BF Cell Signaling Dimethyl-Histone H3 (Lys36) (C75H12) Rabbit 2901BFCell Signaling Dimethyl-Histone H3 (Lys27) Rabbit 9755BF Cell SignalingDimethyl-Histone H3 (Lys79) Rabbit 9757BF Cell Signaling Acetyl-histoneH3 (Lys9) (C5B11) Rabbit 9649BF Cell Signaling Acetyl-histone H3 (Lys14)Rabbit 4318BF Cell Signaling Acetyl-histone H3 (Lys27) Rabbit 4353BFCell Signaling Acetyl-histone H3 (Lys56) Rabbit 4243BF Cell SignalingAcetyl-histone H3 (Lys18) Rabbit 9675BF Cell Signaling LC3A (D50G8)Rabbit 4599BF Cell Signaling LC3B (D11) Rabbit 3868BF Cell Signalingp21waf1/cip1 Rabbit 2947BF Cell Signaling Beclin-1 (D40C5) Rabbit 3495BFCell Signaling β-Catenin (6B3) Rabbit 9582BF Cell Signaling Slug (C19G7)Rabbit 9585BF Cell Signaling Snail (C15D3) Rabbit 3897BF Cell SignalingTCF8/ZEB1 (D80D3) Rabbit 3396BF Cell Signaling c-Myc (D84C12) Rabbit5605BF Cell Signaling Met (D1C2) Rabbit 8198BF Cell SignalingPhospho-Src family (Tyr416) Rabbit 6943BF Cell Signaling Phospho-Jak2(Tyr1007) Rabbit 4406BF Cell Signaling Phospho-Jak3 (Tyr980/981) Rabbit5031BF Cell Signaling Phospho-PLCγ1 (Tyr783) Rabbit 2821BF CellSignaling Bcl-2 (D55G8) Rabbit 4223BF Cell Signaling Bcl-xL (54H6)rabbit mAb #2764 Rabbit 2764BF Cell Signaling Control mouse IgG1 Mouse400102 Biolegend Control mouse IgG2a Mouse 400202 Biolegend Controlmouse IgG2b Mouse 401202 Biolegend Control rabbit Rabbit 550875 BDBioscience Control rat IgG2b Rat 553986 BD Bioscience Her2 Human/MouseHerceptin Genentech EGFR Human/Mouse Cetuximab Bristol-Meyers EpCAMMouse MAB9601 R&D MUC1 Mouse M01102909 Fitzgerald MUC16 Mouse ab1107abcam EpHA2 Mouse MAB3035 R&D FOLR1 Mouse MAB5646 R&D FSHR MouseGTX71451 Genetex TSPAN8 Mouse MAB4734 R&D Claudin-3 Mouse MAB4620 R&DTransferin Mouse MAB2474 R&D CD44s Mouse BBA10 R&D CD44 Mouse 103002Biolegend E-Cadherin Mouse 324102 Biolegend CEA 10-C10C Fitzgerald B7-H3MAB1027 R&D EMMPRIN Mouse MAB972 R&D CD45 Mouse 304002 BiolegendCalretinin Mouse sc-135853 Santa Cruz biotechnology Ki67 Mouse 556003 BDBioscience Control mouse IgG Mouse 5414BF Cell Signaling Control rabbitIgG Rabbit 3900BF Cell Signaling

The antibody-DNA conjugates were first evaluated in MDA-MB-231 (humanbreast cancer) cells. Cells were blocked to prevent nonspecific DNA orantibody labeling and then “stained” with the pooled cocktail followingtechniques akin to standard flow cytometry staining known in the art.Next, DNA was released with a light pulse, hybridized to fluorescentbarcodes, and imaged on a cartridge via a charge-coupled imaging device(CCD) (NanoString Technologies).

Several DNA conjugation using various cleavable linkers andcorresponding release methods were evaluated and optimized (FIGS. 2 and3) Among the tested cleavable linkers, the photo-cleavable linker wasselected for its superior performance (FIG. 2). Probe quantificationtranslated into proteomic sample profiling (FIG. 1C) by normalizingaccording to DNA per antibody and housekeeping proteins (FIG. 4). Onaverage, there were about three to five DNA fragments per antibody;markers were thresholded on the basis of nonspecific binding of IgGcontrols.

Repeated analyses showed consistent results across different batches ofcells analyzed on different days and over time (FIG. 5). In subsequentstudies, antibodies that did not fall above 1.2-fold control IgGthreshold were not included [for example, dimethyl-histone H3 (Lys⁴)].Excluding these outliers, the median SE across all antibodies was 6%. Aprofile of the human MDA-MB-231 line was derived from about 50 cells andshowed, for example, high expression of keratin 7 and epidermal growthfactor receptor (EGFR), two diagnostic markers commonly used inpathology laboratories to identify cancer subtypes. Epigenetic andphosphoproteomic markers have lower expression because these naturallyoccur at lower abundance in cells relative to extracellular markers.Intracellular markers such as phospho-Src (pSrc) and phospho-glycogensynthase kinase 3β (pGSK3β) could also be detected, e.g., using theoptimized permeabilization method (FIGS. 6A-6B).

Additional benchmarking experiments were performed to demonstrate assayconsistency and reproducibility. Conjugated antibodies behaved similarlyto native, unmodified antibodies as evidenced by head-to-head comparisonon flow cytometry (FIG. 7A). Similar results were found when testingintracellular antibodies such as p53 and phospho-S6 ribosomal protein(pS6RP) with dot blots and immunoblotting (FIG. 7B). Antibody-DNAconjugates generated equal or stronger signals compared to nativeantibodies on dot blots. Furthermore, the DNA-modified antibodies showedsimilar expression patterns across cell lysates when compared to nativeantibody. To assess reproducibility, two DNA-modified antibody clonesspecific to the same target [e.g., epithelial cell adhesion molecule(EpCAM)] were shown to give nearly identical expression levels (R²=0.99)across multiple cell lines and clinical samples (FIG. 8A). Antibodystaining was evaluated using both a cocktail of 60+ antibodies and assingle agents; expression levels from both methods, as measured by anantibody barcoding with photocleavable DNA (ABCD) platform as describedherein, showed high, linear correlation (R²=0.93; FIG. 8B). Proteinmarker changes measured with the ABCD platform linearly correlated toexpression changes measured by independent immunofluorescence studies intaxol-treated HT1080 fibrosarcoma cells (FIG. 8C). Flow cytometrymeasurements across eight cell lines and six different markers showedlinear correlations (R²=0.92 to 0.99) (FIG. 9).

Example 3. Single-cell Sensitivity of One Embodiment of the Methods forMultiplex Detection of Target Molecules from a Sample (AntibodyBarcoding with Photocleavable DNA (ABCD) Platform)

The sensitivity of the ABCD platform was assessed by detecting acrossvarying cell numbers (50, 15, 10 or 5 cells) from a bulk sample of500,000 cells, in multiple repeats, by serial dilution (FIG. 10A). Thecorrelations between bulk and diluted DNA counts were linear, withcorrelation coefficients >0.9 (FIG. 10B). Additional experiments wereperformed to validate the ABCD platform in single human A431 cells. FIG.10C displays the expression levels of 90 analyzed proteins for fourrandomly chosen single cells and in bulk samples. Consistent withprevious reports (13), there were some intercellular heterogeneity, butgenerally, single-cell profiles matched their respective bulk profileswith correlations as high as 0.96 and as low as 0.63. Multinucleatedcells were excluded; cells were otherwise selected at random.

To demonstrate biological variation at the single-cell level, untreatedsingle human A431 cells were compared to cells treated with gefitinib-aselective tyrosine kinase inhibitor of the EGFR. Unsupervised clusteringof single cells showed unique patterns for treated and untreated groups(FIG. 11A). A431 cell lines overexpress EGFR and are highly sensitive togefitinib [median inhibitory concentration (IC₅₀)=100 nM], as shown bywidespread pathway inhibition in gefitinib-treated A431 cells. Athreshold was applied at the single-cell level to ensure that markerexpression levels were detectable above all six IgG controls for allcell lines. The majority of the panel was still detectable, althoughsome markers such as phospho-EGFR fell below threshold levels in somecells, and thus were not included for hierarchical clustering.Nevertheless, pairwise comparisons between the two cohorts showedsignificant changes in key markers (FIG. 11B) such as pS6RP, Ku80, andphospho-histone H3 (pH3). These changes in the markers were alsoconsistent with previous reports (14, 15). Unlike most signalinginhibition studies, the untreated cell line was not prestimulated withEGF before treatment. Hence, the assay conditions mimicked naturalsignaling variability to better approximate patient samples.

Example 4. Measuring Inter- and Intratumoral Heterogeneity in ClinicalSamples Using the ABCD Platform

To demonstrate the clinical capabilities of ABCD and explore single-cellheterogeneity, FNAs were obtained from patients with lungadenocarcinoma. Single-pass FNA samples were initially processed usingantibody-mediated magnetic selection to isolate EpCAM-positive cells.Single cells for subsequent analyses were harvested viamicromanipulation, whereas other sample debris was removed. In onerepresentative patient, protein marker expression in 11 single cells(EpCAM+/DAPI+/CD45-) correlated with bulk measurement (about 100remaining cells from FNAs) (FIG. 12A). Yet overall, correlation betweenpatient cells and bulk FNAs was lower and varied compared to singlecells from cell lines and their respective bulk in FIGS. 10A-10C. Thehighest correlation with the bulk measurement was 0.79 (cell cultureshowed R=0.96), whereas the lowest value was 0.43 (FIG. 12B).

Interpatient heterogeneity in bulk samples was next determined from sixpatients with biopsy-proven lung adenocarcinoma (FIG. 13). Althoughthese cancers harbored identical histopathology, proteomic profilingrevealed clear differences, even in this small cohort. Marker panelswere chosen to evaluate protein heterogeneity across a broad range offunctional protein networks (16) relevant for therapy assessment. FIG.13 shows visual similarity among patients 1, 2, and 5 (SpearmanR_(1,2)=0.94, R_(1,5)=0.96, R_(2,5)=0.95). This partially concurred withgenotyping because both patients 1 and 2 had EGFR T790M mutations,whereas patient 5 had a KRAS mutation (KRAS 35G>T). This indicates thatdifferent genotypes may still yield similar proteomic phenotypes.Patients 3, 4, and 6 harbored distinct proteomic profiles and differingmutations (FIG. 13). Patient 3 had an exon 20 EGFR mutation, whereaspatient 4 had an EGFR L858R mutation and an additional BRAF mutation.Patient 6 was noted to have an EML4-ALK translocation.

Protein clustering also showed possible personalized targets (FIG. 13).For example, patient 4 (EGFR/BRAF mutant) had high phospho-extracellularsignal-regulated kinase 1/2 (pERK1/2) and pS6RP, as expected for apatient with an EGFR L588R mutation; however, this patient also showed ahigh level of the DNA repair/damage markers poly(adenosinediphosphate-ribose) polymerase (PARP), Ku80, and phospho-histone H2A.X(pH2A.X) expression, indicating that PARP inhibitors or DNA-damagingagents (for example, cisplatin) could be effective for this patient.Thus, such information determined by methods for detecting a pluralityof target molecules as described herein (e.g., ABCD platform) can beused to complement pharmacogenomics.

Example 5. In Vitro Discrimination of Pathway Analyses during TreatmentUsing ABCD Platform

Having established feasibility of inter- and intrapatient analyses inclinical samples, it was next sought to explore the feasibility ofmonitoring cancer treatment over time. To this end, it was first soughtto determine if known pathway responses to different drug treatmentscould be discriminated. FIG. 14A shows the validation thattriple-negative breast cancer cells (MDA-MB-436) treated with kinaseinhibitors (gefitinib and PKI-587), antibody drugs (cetuximab), andDNA-damaging drugs (olaparib and cisplatin) showed profiles thatclustered according to drug mechanism of action. As a control study,cell lines treated with cetuximab resulted in expected drug inhibition(FIG. 15B). Expected protein inhibition in drug-sensitive human cancercell lines using optimized drug doses and incubation times wasdemonstrated using the ABCD platform. Notable examples include pS6RP fortargeted treatments, and pH2A.X, pATM/ATR (phospho-ataxia telangiectasiamutated/ATM- and Rad3-related) substrate, and cleaved PARP forDNA-damaging agents. Unexpected results, such as epigenetic histonemodifications after treatment with a phosphatidylinositol 3-kinaseinhibitor (PI3Ki) was also found (FIG. 15E). For additional in vitrovalidation of treatment, HT1080 fibrosarcoma cell lines were treatedwith four different doses of taxol. Several panel markers displayeddose-response changes to taxol treatment, including pERK andphospho-cyclin D.

Proteomic profiling of olaparib and cisplatin treatments was performedfor four human cancer cell lines, showing varying drug sensitivities asmeasured by viability assays (FIGS. 14A-14B and FIG. 15A). The degree ofchange in protein profiles was quantified by calculating the number ofmarkers that were significantly different from the untreated conditionusing pairwise t test [false discovery rate (FDR)=0.1]. This profilingindicated that global pharmacodynamic changes correlated with treatmentsensitivity: As IC₅₀ values decreased, the number of protein markerswith significant changes increased (FIG. 14B). For resistant cell lines(for example, OVCA429), no significant changes were detected. Expectedchanges in DNA damage and apoptosis markers, such as degradation of Bimand up-regulation of pERK (FIGS. 15C-15D) were also detected, indicatingprevious studies of DNA damage response to cisplatin treatment (17).

To evaluate the assay's ability to measure even small marker changes,HT1080 human fibrosarcoma cells were treated with taxol at fivedifferent doses. Marker changes at high doses were compared to markerchanges quantified by an independent immunofluorescence screen (FIG.16A). Several protein markers showed dose-response curves, includingCDCP1, phospho-cyclin D, cyclin El, fibroblast growth factor 4 (FGF4),BRCA2, and pERK1/2. These in vitro studies established that the markerpanel could indeed measure pathway changes in response to varying drugmechanisms; furthermore, these changes could be detected in a sensitive,dose-dependent manner. Additionally, pairwise t tests between the dosedand untreated cells showed an increase in significant marker changes atthe highest dose (700 nM taxol) compared to the lower 70 nM dose (FIGS.16B and 16C).

Example 6. Monitoring PI3Ki Treatment Response in Cancer Patients

In some embodiments, it is desirable to translate these pathway analysesto patient samples, e.g., to analyze serial biopsies in early-phaseclinical trials with the goal to better assess drug efficacy and dosage.However, such invasive procedures can introduce risk of morbidity andhigh costs. The ability to analyze small numbers of cells fromalternative sources (for example, FNAs) becomes paramount whenresponsive tumors shrink after treatment, making repeat biopsiesdifficult. As proof of concept, scant cell analyses were performed infour patients before and after PI3Ki treatment during phase 1 doseescalation trials (FIG. 17A). Pretreatment samples were collected theday before the first drug dose; post treatment samples were collected atthe end of the second treatment cycle. Collection and processingoccurred over the course of a year to correlate profiles to patientresponse. All four patients had metastatic cancers of various subtypesand were selected on the basis of genetic PI3K mutations that couldpredispose their tumors to pathway inhibition using PI3Ki treatment. Inall, two patients responded and two progressed. Data analysis wasperformed in a blinded manner. Unsupervised clustering separated out twogroups of responders versus non-responders (FIG. 17A). Among the tworesponders, one patient showed larger fold changes across the markerpanel. Subsequent unblinding revealed that this patient received ahigher dose of the drug during phase 1 dose escalation than did theother responding patient. Additional patient samples can be used tomeasure ABCD platform's clinical impact during drug dosing pathwaystudies.

In some embodiments, the screen performed by the ABCD platform couldhelp predict clinical outcome or identify promising markers of treatmentresponse. To demonstrate this, five drug-naïve patients, all withvarious PI3K mutations, who eventually received small-molecule PI3Kitreatment were profiled. Patients were categorized as nonresponders orresponders (FIG. 17B) and a marker-ranking algorithm was used todetermine top differential markers. The top marker, di-methylation ofhistone H3 at Lys79 (H3K79me2), clustered with several markers: pS6RP (aknown downstream target of PI3K and an emerging key biomarker oftreatment response) (14), pH2A.X, and PARP. According to canonicalpathway signaling, selecting epigenetic or DNA damage markers asreadouts of PI3K treatment response would not be an intuitive decision.DNA damage and epigenetic marker changes were also identified by invitro profiling of a PI3Ki (FIGS. 15C-15E). This cluster covered diverseproteins across various pathways: epigenetic changes, DNA damage, andgrowth and survival pathways [PI3K and mitogen-activated protein kinase(MAPK)], indicating the potential value of system-wide profiling fordeveloping better companion diagnostics during treatment.

Discussion Based on Examples 1-6

In some embodiments, presented herein is an amplification-free methodcapable of sensing hundreds of proteins in human cells by using one ormore embodiments of target probes described herein (e.g., DNA-barcodedantibodies) coupled with highly sensitive optical readouts. Celllabeling, washing, and analysis can be completed within hours, makingsame-day protein analysis possible. The method measures more markers onlimited material than immunohistochemistry and preserves geneticmaterial from samples, which is not possible with traditional tools likemultiplexed cytometry (18). The protein coverage and/or methodsdescribed herein can be extended to include additional protein targetsand/or other target molecules through conjugation of target molecules toidentification nucleotide sequences (e.g., antibody-DNA conjugations),resulting in a scalable, multiplexed target molecule (e.g., protein)screening platform.

In general, the method can provide analyses of protein expression levelsfor both single and bulk cell populations. The in vitro studies as shownin the Examples showed that single cells from cell lines showed highercorrelation to bulk measurements than those isolated from patienttumors. In FNAs, the single cells also showed higher correlations witheach other than with the bulk population. This could be, for example,because an averaged bulk measurement is less likely to correlatestrongly with a single clonal phenotype.

The findings presented herein showed that the methods described hereincan be used to detect extracellular proteins (e.g., but not limited to,CD44, EGFR), intracellular/cytosolic proteins (e.g., but not limited to,p-S6RP), and/or intracellular/nuclear proteins (e.g., 53BP1) in asample.

The findings presented herein also showed that current cell culturemodels are an insufficient estimate of proteomic heterogeneity inclinical samples. The methods for detecting a plurality of targetmolecules from a sample described herein (e.g., ABCD platform tool) aretherefore useful for its ability to study rare single cells in clinicalsamples, such as circulating tumor cells, stem cells, and immune cellpopulations. As shown herein, even scarce proteins, such as 53BP1 andpH2A.X, could be detected at the single-cell level. Large-scale proteinmapping of isolated, rare cells and clonal populations could shedinsight into cancer heterogeneity, drug resistance, and the clinicalutility of circulating tumor cells. Intratumoral heterogeneity mayitself be a biomarker of poor clinical outcome (19). Thus, the methodsdescribed herein (e.g., ABCD platform) can be used to determineintratumoral heterogeneity, which can be used as a biomarker fordiagnosis and/or prognosis. Establishing causal and reactivecorrelations between diseases and altered biomarkers could alsoradically improve physicians' abilities to diagnose and treat patients(20, 21). In some embodiments, the methods described herein (e.g., ABCDplatform) can be used to determine causal and reactive correlationsbetween diseases and altered biomarkers in order to improve physicians'abilities to diagnose and/or treat patients.

The inventors have demonstrated the ABCD method's ease of use,reproducibility, compatibility with clinical applications, such asprofiling of FNA cancer samples, and its translational potential tomonitor cancer treatment as demonstrated in four patients. The findingsshowed that broader profiling can improve understanding aboutpotentially useful companion diagnostic biomarkers and help explore howdrug dosing corresponds to cellular pharmacodynamics. Smarter proteinmarker selection, as demonstrated by the ABCD platform, could markedlyreduce drug development costs, narrow patient cohorts, and improveclinical trial design.

The methods described herein (e.g., ABCD platform) could complementother art-recognized single-cell proteomic techniques, such as masscytometry and fluorophore-inactivated multiplexed immunofluorescence (8,22). One of the advantages of the methods described herein (e.g., ABCDplatform) is that both genetic material and protein barcodes can beconcurrently extracted from a single sample, thus paving the way formore biologically relevant analyses of protein-DNA-RNAinterrelationships. Such integrative measurements could explain “missingpieces” in genomics associated with various diseases or disorders, e.g.,cancer genomics. For example, in the Examples presented herein, not allpatients with PIK3CA DNA mutations responded to a given PI3Ki; this isconsistent with clinical experience (23, 24). However, proteomicbiomarkers revealed differential changes between responding andnonresponding cohorts. The Examples indicate that protein profiling willhelp complement genotyping to shape therapeutic advances for cancer andother diseases.

The Examples presented herein demonstrated proof of principle that thetechnology described herein can work in clinical samples with a widerange of applications, including rare cell profiling and companiondiagnostics within cancer clinical trials.

In some embodiments, the technology described herein (e.g., ABCDplatform) can be modified to suit the needs of various applications. Forexample, the methods described herein (e.g., ABCD platform) can beadapted to work with both whole cells and/or cell lysates, and DNA canbe quantified with other readouts (for example, sequencing) to performsimultaneous measurement of RNA, DNA, epigenetic, and proteinexpression. In some embodiments, the methods described herein (e.g.,ABCD platform) can include a module to rapidly isolate and measureentire populations of single cells. For example, additional componentsand wells can be added to microfluidic devices such as the one describedin the Examples to increase the throughput of single-cell analysis.

Single-cell studies can be validated with a higher-throughput device.For example, larger numbers of cells can be used to compare populationdifferences and spreads between the methods described herein and othergold standards (for example, flow cytometry). In some embodiments, themethods described herein (e.g., ABCD platform) can be used to identifynovel companion diagnostic markers or specific pathway markers fordiagnosis of a disease or disorder (e.g., cancer subtypes) and/ormonitoring patients' response therapeutics.

The methods described herein (e.g., ABCD platform) can enablelarger-scale studies to yield mechanistic insights into existing and/ornovel therapeutic strategies. Moreover, the methods described herein(e.g., ABCD platform) can also be used for rare, single-cell (forexample, but not limited to circulating tumor cells) profiling to derivefurther understanding of their biological and clinical relevance.Because genetic material from samples is preserved, the methodsdescribed herein (e.g., ABCD platform) can be adapted to study proteinsthat interact with genetic regulatory elements such as microRNAs. Themethods described herein (e.g., ABCD platform) can be used for variousapplications in research laboratories, academic hospitals, andpharmaceutical companies to help propel drug trials and biologicalinvestigation.

Exemplary Materials and Methods for Examples 1-6

Study Design. In order to determine if protein networks (as opposed tosingle biomarkers) will reveal clinical or biological insights into howa disease or condition (e.g., cancers) evolves and responds to drugs, amultiplexed platform for detecting protein expression, e.g., in clinicalsamples and in cell lines, was developed. The Examples hereindemonstrate the use of the methods described herein (e.g., ABCDplatform) in understanding treatment response in cancer

Clinical studies were performed on limited cohorts of patients for proofof principle. The number of patients was selected based on a 1-yearenrollment cycle (March 2012 to March 2013). All protein measurementswere included as long as their signals were above a pre-determinedthreshold. In one embodiment, the threshold was ˜1.2-fold higher thanthat of its corresponding nonspecific IgG isotype. This threshold wasset to be over three times the median SE from the antibody cohortspooled. Only antibodies that were validated (via flow cytometrymeasurements on cell lines) were included. All in vitro studies wereperformed in replicates (n=3, unless otherwise specified). Afteroptimization, studies with the final protocol were repeated multipletimes on different days to ensure consistency and reproducibility. Allexperiments on clinical studies were performed blinded duringexperimental procedures and raw data analysis.

Cell lines. Validation experiments were performed in the following celllines, which were purchased from the American Type Culture Collection(ATCC): SKOV3, ES-2, OVCA429, UCI-107, UCI-101, TOV-112D, TOV-21G,A2780, MDA-MB-231, MDA-MB-436, A431, and HT1080. Cells were passaged inDulbecco's modified Eagle's medium (Cellgro) or RPMI (Cellgro) asrecommended by ATCC. cell lines were derived from ovarian surfaceepithelium (OSE) brushings cultured in 1:1 Medium 199/MCDB 105(Sigma-Aldrich) with gentamicin (25 mg/ml) and 15% heat-inactivatedserum. TIOSE6 cell lines were obtained by transfecting hTERT into NOSEcells maintained in 1:1 Medium 199/MCDB 105 with gentamicin (25 mg/ml),15% heat-inactivated serum, and G418 (500 mg/ml) (25). Aftertrypsinization, cells were immediately fixed with 1× Lyse/Fix buffer (BDBioscience) for 10 min at 37° C. and then washed twice withSB+[phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA)].The cells were aliquoted into tubes (˜1×10⁶ cells/ml) and stored at −20°C. until labeling. Biological replicates were seeded in different wellsand collected separately. Cultured cells were processed and stored underthe exact same conditions as clinical samples. A total of 276 sampleswere prepared and analyzed independently via the barcoding method.

Clinical samples. The study was approved by the Institutional ReviewBoard at the Dana-Farber/Harvard Cancer Center, and informed consent wasobtained from all subjects (n=10). Fourteen minimally invasiveprocedures were performed on the 10 enrolled patients. Six patients hadprimary lung adenocarcinomas. The four patients undergoing PI3Kitreatment with repeated biopsies had carcinomas of varying origins inthe abdomen, all with underlying PI3K mutations. All pretreatmentbiopsies were collected in the week before the first cycle of treatment.All post treatment biopsies were collected after a cycle was completed,typically after several weeks to months. Image-guided FNAs with a22-gauge needle were obtained before routine core biopsies. Correctneedle location was confirmed by computed tomography imaging andreal-time readout by cytopathology. FNA samples were processedimmediately by centrifugation and removal of excess PBS. If there werevisual clumps present before the fixation step, collagenase(Sigma-Aldrich) was added at 0.2 mg/ml. Cells were fixed with Lyse/Fixbuffer (BD Biosciences) for 10 min at 37° C. and washed twice with PBSwith 2% BSA. All centrifugations were performed at 300 g for 5 min.Clinical samples were stored at −20° C. A total of 24 samples wereprepared and analyzed independently via the barcoding method.

Drug treatments of cell lines. To test the effect of drug treatment onprotein expression levels, the cell lines were treated with a number ofdifferent chemotherapeutic or molecularly targeted drugs. A431 celllines were dosed with gefitinib (Selleck Chemicals) in medium with 1%dimethyl sulfoxide (DMSO) for 12 hours at a concentration of 10 μM. Thetriple-negative human breast cancer MDA-MB-436 cell line was dosed withthe PARP inhibitor olaparib (10 μM in 0.1% DMSO in medium), cisplatin(10 μM, 1% Hanks' balanced salt solution in medium), the PI3K/mTORinhibitor PKI-587 (100 nM, 0.1% DMSO/medium), and the EGFR inhibitorscetuximab (75 μg/ml in medium) and gefitinib (10 μM in 0.1%DMSO/medium). All molecularly targeted agents (PKI-587, cetuximab, andgefitinib) were applied for 12 hours. DNA-damaging agents olaparib andcisplatin were applied to cells for 3 days. Changes in proteinexpression levels were compared to medium controls under identicalconditions but without drug treatment.

Flow cytometry. Flow cytometry was used to validate protein expressionlevels in bulk samples. Fixed cells stored at −20° C. were thawed andthen permeabilized with a saponin-based buffer, PW+(1× Perm/WashPhosflow Buffer, BD Biosciences, with 2% BSA). About 200,000 cells pertube were incubated with primary antibodies for 1 hour at either 1 mg/mlor the appropriate dilution as recommended by Cell Signaling for flowcytometry applications. An example list of primary antibodies is shownin Table 1 above. After one wash with PW+, the appropriate secondaryantibodies targeting mouse, human, or rabbit IgG were applied. Thespecific secondary antibodies used were anti-rabbit IgG (H+L) F(ab′)₂Fragment Alexa Fluor 647 Conjugate (Cell Signaling #4414), anti-mouseIgG (H+L) F(ab′)₂ Fragment Alexa Fluor 647 (Cell Signaling #4410), andanti-human FITC (Abcam ab98623). Expression levels for each protein werethen calculated by normalizing the geometric mean from each antibodywith the appropriate control IgG. These values were then correlated tothe expression values derived from the DNA barcoding technique.

Synthesis of photocleavable DNA-antibody bifunctional linker. Thephotocleavable linker was synthesized as previously described in Ref. 9.For example, compound 1 (FIG. 2B, ˜0.100 g, 0.334 mmol) was dispersed in5 ml of dry dichloromethane (DCM) in a round bottom flask under argonatmosphere. The flask was cooled to 0° C. by placing it on an ice bath.2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) (0.139 g, 0.368 mmol) and triethylamine (TEA) (109 0.835 mmol)were added to the solution. The reaction mixture was stirred at 0° C.for 5 min, and N-(2-aminoethyl)maleimide trifluoroacetate salt (0.093mg, 0.368 mmol) was subsequently added. After stirring at 0° C. for 15min, the reaction mixture was allowed to equilibrate to room temperaturewhile being stirred for 18 h. After the reaction mixture was dilutedwith DCM (45 ml), the organic phase was washed with water and saturatedNaCl solution, then dried over sodium sulfate. The organic layer wasconcentrated under reduced pressure and charged to a SiO₂ column(eluent: 100% DCM to 3% methanol in DCM, v/v) for purification. Theyield of compound 2 was approximately 60%. ¹H NMR (400 MHz, CD₃OD): 7.58(s, 1H), 7.37 (s, 1H), 6.77 (s, 2H), 5.44 (q, ⁴J=6 Hz, 1H), 4.03 (t,³J=6.4 Hz, 2H), 3.94 (s, 3H), 3.61 (t, 3J=5.6 Hz, 2H), 3.35 (t, 2H,overlapping with the solvent residual peak), 2.32 (t, ³J=7.2 Hz, 2H),2.05 (m, ³H), 1.46 (d, ²J=6.4 Hz, 3H). MS (electrospray ionization massspectrometry: ESI-MS) calculated: 421.15, found: 466.18 {M+HCOO}⁻.

Compound 2 (0.010 g, 0.024 mmol) was dissolved in anhydrousdimethylformamide (DMF) (1 ml). N,N′-Disuccinimidyl carbonate (DSC;0.018 mg, 0.071 mmol) and TEA (12.5 0.096 mmol) were successively addedto the solution. The reaction mixture was stirred at RT for 18 h. Thereaction mixture was directly loaded onto a C18 reverse phase column forpurification (eluent: 5% acetonitrile in water to 95% acetonitrile inwater, v/v). The yield of the photocleavable bifunctional linker productwas approximately 70%. ¹H NMR (400 MHz, CDCl₃): 7.63 (s, 1H), 7.05 (s,1H), 6.67 (s, 2H), 6.48 (q, ⁴J=6.4 Hz, 1H), 6.03 (br, 1H), 4.08 (t,³J=5.8 Hz, 2H), 4.02 (s, 3H), 3.68 (m, 2H), 3.45 (m, 2H), 2.79 (s, 4H),2.36 (t, 3J=7 Hz, 2H), 2.15 (m, 3H), 1.75 (d, ²J=6.4 Hz, 3H). ESI-MScalculated: 562.15, found: 607.22 {M+HCOO}⁻.

DNA-antibody conjugations. Antibodies (e.g., listed in Table 1) wereconjugated to specially designed alien DNA sequences derived from thepotato genome (exemplary sequences shown in Table 2).

TABLE 2List of example 70 mer alien sequences used for barcoding a target-binding moleculeT_(m) T_(m) capture reporter Target sequence probe (° C.) probe (° C.)GCTAAGTTTGGAATTAAGAAAGGAGTTGCTGGAGGTCCTTTCCAGCATAAG 79 79AACCAGCCATATTGCTTAA (SEQ ID NO: 1)TGCCTTCTGAAAGAGACGTTATTGTTGAAGCAAGAGATAGCTTAGTAACAA 80 78ATGCTATAGCTCAGGCAGG (SEQ ID NO: 2)CCTGATCATGCTTTGTCAGCAGACCCAGAAGAATTCATCACAATCACTGGA 82 78AGATTGAGCTTAGGAAAGT (SEQ ID NO: 3)GAGCGGATGTTATTGAGAAGCACTTTACCTTAGATTTCTAAAGCTCTCTTCC 78 82TCCTCTCTTCTCCGCTCA (SEQ ID NO: 4)ATCGGCTGTGCGATTGCTATTGATGTGTTAAGAAATTTGGTTTGTGATTGGC 80 81AAATCTCTCCTCCAACTC (SEQ ID NO: 5)ATTTGGATGAAGTCGGCTTTATGGTGACACAAATCATGATGAGCTGAGGTT 79 82CTGACAGCAAATACGCTCA (SEQ ID NO: 6)ATAGAACCATTTGCTGATGAGGTGACAACAGATCGTTGCACTTATGCTATC 80 78CCGTTAGACTATCTGCTAT (SEQ ID NO: 7)ACTACCATGTACTGCGCGAGACTAGCCTATCATTGGATTGCAGCGATGACT 82 81ATATCTGAGCACCTGTGAC (SEQ ID NO: 8)ATATGAGACGACTAGCACGCCATAGCGTTACATACGTGTCGATCCGAGAAC 80 81ATCACTCTAATGACGAGTG (SEQ ID NO: 9)CATCATCGACAGTTCGCAGCCCTATAACATGATACTAGATAACGATGCTCC 80 79ATGTTAGTGAATGCGAGTC (SEQ ID NO: 10)ACTCACACATAGTACTGACACGTAAGATAGGATGCTATATGGTCATTGGTC 79 79ACCCGAGTTACGATCAAAT (SEQ ID NO: 11)CAGATAGACTCACCTCGATATACAGGGAGCCACGACTTAGGACTATGGATA 82 78AGTCATCTAAAGCGTCCGA (SEQ ID NO: 12)CACTGTCTATACATGGACGACACTTTGCACATCATTACCAAAGAGCGCAAC 80 81GTATCTAGGATTGAGCAGT (SEQ ID NO: 13)AGACTAATTGATCGGACCGATGACAGTTCACAGAGGGATACACTGTTGAGC 80 80CGACCCTATTAGCTGATAT (SEQ ID NO: 14)TGATCCACACTGACGAATCATGTACTCACTCGATCGCCACTTCACACAAGA 80 79ACACAAATTTGGAGTATTG (SEQ ID NO: 15)CTCGAGAATCACACACAGTCGTCTAAGACACGACAAGTGCAACAGCAATC 81 78CACATCTTAGATGAGATTAG (SEQ ID NO: 16)CGATTACAAGGCGTGGTCAGATATTAGACTCCAGGGGATTTAATGCCAGTC 81 81CAAGCTCTCTTCCACATTC (SEQ ID NO: 17)ATCTGCATGAACGGGAAAGGAGTTCGATGAGACTTTCAAACCAACATAATG 82 80TCTCTCCAACCTCAGGAAG (SEQ ID NO: 18)ATAGTCTTTAGAGCCTCAGAATAGGCTGTGACGCGGAAGATAACTCATAAG 82 79TGCCTCCCTCGGTAATTTG (SEQ ID NO: 19)GCCAGGTATGCCGTGAACGAGTTCTTCATTAACTGTTATGTCTCGGGAGTCT 82 80GATATTGGTACTTCTCCC (SEQ ID NO: 20)TTAGCACCGATATCAATACTGATGATGTCACCGTCGAGCTCGTGTTGAACC 79 82CTTCAAGTAACAACCTGAC (SEQ ID NO: 21)ACTTGTTCGACTGACAGTTTAACGCCTGACATGAACGGCTTGCTTATAATGA 81 81CTGGCAGGGTTATGAATG (SEQ ID NO: 22)AAACTGACCGTACCGTTAGAAGAGAGTTCCGCTTCTCTCATGATGTGCGCA 82 81TCTCCCACATTATTTGACC (SEQ ID NO: 23)TGATGACAGTGACAATTGACCGAATTGCCTGATCATTACCTTACAGTGCGC 81 79AGATTGGGATAATCGATTT (SEQ ID NO: 24)TAGGCGTTGAGGCTTTGTTTCTTTGCCTCTATTGTAAGACTCATTCTGACGG 81 80CCTCTAGTCGTTGATATG (SEQ ID NO: 25)AAGGACATTCTTTCGAATGCAAGTTCAAGGCACATTTTCTATATCAGCCAC 80 79CATGGGAGTGACATTTCTT (SEQ ID NO: 26)CAATAGCTCCAGTAGTAATTGTTGTCGCTCCGCTGAGCAGTTAATCCTTATG 82 78TCAACAACCTCAGCATAG (SEQ ID NO: 27)TTCACCAAGCTGAACAGGGTTGCGCTGAATAAATTTTACAGGATACTATGG 82 79ACAGGTTCAGAATCCTCGA (SEQ ID NO: 28)GGAATGAATCCATTGCATTTCCATGAGAATGCAGACTTAATCGGACGTATC 79 80GACTTTGGGTCCACGATAT (SEQ ID NO: 29)GAGGTCTTGTTTCATCTAAACCGAGCAGGATGATAAGCCATAATTCGTAAC 79 79CCGAGGGTATAATTCGTTA (SEQ ID NO: 30)GTCCTTCTGCTTATGACATTCCGTGCATTCCGTAGCTACGTCAAGCGTTACA 82 80TAGTGACGGAACTGTTAG (SEQ ID NO: 31)TCTGTACCTTGGCACTCCATCTGGTAAGTCACTTATAGTTGTATGGTTTCAG 81 80ATGAGGGAACGTGTAGGA (SEQ ID NO: 32)AATTTCTGAGATTGTTGGTAGAGGGAGAAATGGGAAGGACATGTTTCAACA 79 80ATCACCGGATTAAAGCCTT (SEQ ID NO: 33)TGTGGAAGGACTGTGATAAACCAATAGGGTGTCAAGATCTGTAAGTATGGG 80 80ATTAGGGATGTTCTGCCAG (SEQ ID NO: 34)GCCGTCGGACATAACCACTTGGATATATACGTAGTTCATCAACCTTAACTC 80 82CCTCTGGGTTCATTGGGAG (SEQ ID NO: 35)GCTATTGCAGCAAAGAGAACAGACGCTTTAACTGGTATCGAGCGCTTAGAT 81 78GGCTATATGGTCTACTAGA (SEQ ID NO: 36)GAAATCAGATCAGTTCTACATTCGGTGGGAGCCCTCTATATGATTAGATCCT 82 80GCAGCCGTACTTCCGTCA (SEQ ID NO: 37)GGTGGCTTGATTTAACTGAATCAGGCCCTAACCATTTGTATTGTGTCTACAC 82 81TGGTCCGTTCTTAGACGC (SEQ ID NO: 38)GTTGTTTACCTTGTAGATCGACTTCACATCAGCGGCAGAAGGCCCTCAACG 80 81TAAATCTGCTCCACATTTA (SEQ ID NO: 39)TGTTGACATCCGCAACAATGTACCTTATATCGGCATATGGATCTCTTGATCG 81 80AGCGAACCTCCCTTTAAC (SEQ ID NO: 40)AAGGTGATTCACTAACCAGCTCTTACTCCTCGTTCGGTAGCAAATGAAATG 80 81CCGGATGCTGTTGAAGTAG (SEQ ID NO: 41)CGCATAACTCGAACCACAGTTACTATCAGTCGACATCCCACCAGAGAAATT 80 79GAAGGATATTGTTGAAGCA (SEQ ID NO: 42)GAATCTTGGAAGGTTTCCAGTTAAATAGGGCGTGCGAAGATTCCAGGCAGA 81 80TTTCTCAGGAATTCAGTCA (SEQ ID NO: 43)CTGCTAATGCTGATGGCCCACCTTCTCTATTTGTCGCCATTATATGCGTTGA 82 78GGTTAGTTCAAGCAATAC (SEQ ID NO: 44)GAACAGCTTTCCTTGCTCCCTCTAAATCACCATTTCCATTAGATGAAACCGA 80 78CTTCATTCCAGACTCAAT (SEQ ID NO: 45)AATGCATTTGCCAATGTAGCCATTGTATAACCAGATACACTAGTCCAATGT 79 81CTCAACCAGGGATACCACA (SEQ ID NO: 46)CTCAGAGCTTCAAATCTATCCTCTGGAATCTCTGTATAAGCCCTCGAATACA 79 81ACTTGAGGTATCCCGCAT (SEQ ID NO: 47)CTCTTCTGCCCTACATCACTATCGACTATAGCAACATATCTTTCTCGGGTAA 79 78AGATTAGGCGTCCGATAT (SEQ ID NO: 48)GTAACCGTAGTCGCGCAAACCGTTATATTACGGATATGATCCAAGTTATAT 81 79ACATTAGGACGCGGTTGCT (SEQ ID NO: 49)ATGGTTAGTAAACAGCTTTGATTTCTACATCCGCCTAGCAAACCCATAGTTC 79 81TGCAGTAGATTCACAGCG (SEQ ID NO: 50)TTCAGTTATAATGTGTCCAGCAGAAGCAGGAATTGAATTACCCAAGTTGCA 79 78AGTGGAAGATTTGGAGTTA (SEQ ID NO: 51)TTGCAGAAGCATTCCCAATATGGGTTTCAAGAGTTTAAAGAATGTGGAACA 80 79TTCATGGGAACTGGTGAAG (SEQ ID NO: 52)GCAACAACCTCATCTATACTGTGAATAGTCCCTCCGCTGTCTATATTGGAAC 80 82TGCTGCAATGGTTGCTCT (SEQ ID NO: 53)CCGCAGATTATCGTTTACGATGCATCCATGGTCTCCGACCCATTGAGAGAG 82 80CCAATGGAATTAAGAACTT (SEQ ID NO: 54)CACCATTCAGCCTGATATTGCGTTTGGTGTTGATGTGGCAACTGCATACTGA 81 80ATAACTCCCTGAAATAGC (SEQ ID NO: 55)CGTTACATACTCAGCCATAGGCTTCGATAACAGCATTATTGGAACCTCTGG 81 79GACATTAACAGAGACAACA (SEQ ID NO: 56)AGCGTACTAGGCATCTATTGGCTGAACTACCATGTAATTAGTGGTGTTCCA 81 80GCCTCTAAGATGATGTGGT (SEQ ID NO: 57)GATAGGATGCGACTGCGTATCATATAGGCTGCACATTAGCTGTTGCTTCAA 82 79ATGCCAATCTTACCTCAAC (SEQ ID NO: 58)AATGTATGAGCGGACACTATGCTAAGAGAGACTCCATCAATCCCTCTATGC 80 79AAGATAACAACATCTGGCT (SEQ ID NO: 59)TGCACATCATAGTGCGACGTTGATCCAGATAGACTATAAGACGGCTTGGCA 81 80TTTACCCTAGTCACTATCT (SEQ ID NO: 60)AATGTGTCAGCGGCCTAACTGTAATTGATCCACACCTTAGTTCGGGAGCTA 82 80CCGATCTAATCAACCGTTT (SEQ ID NO: 61)AGACTCCAGGTCGATCATTGGATAACCAACCAGTCGGTTATCCATGACGAG 82 80TGAATAATCTTACCGCAGG (SEQ ID NO: 62)TTTAGATCCTAAGAATGCGAAATGCCGATTCCCGCATATTTCGTAAGCTCGT 82 81TCGGGACTTTGTATCGGC (SEQ ID NO: 63)GAGTGATAGGATCACTCTAAGATCGGCCACTATACGACGCTGAGGTTTATA 79 81TGAACGGCCGCAATTATGA (SEQ ID NO: 64)TCTTGACCAACACCATGTCCGACATACTCCCTAACATGGGTACGGCGACTA 82 82CTGAATCGTTCTTTGAGAG (SEQ ID NO: 65)TGTGTAAATGAAAGCATCTGACTCAACAGGCATCAGTAACGATAATGAGTA 80 79CAACGCCCAATGGTCATAG (SEQ ID NO: 66)GCTTCAACGATTTCAATATACCCATTCGTCAGAGGAAGTAGTAGATCCCGC 79 81CGTCTTAGTCGGATTGAAA (SEQ ID NO: 67)TGTGGTTCCGGTTGCGTATAGATCATGATTCTTTACCCACCTCTTGCTGTAA 79 82TGACCACAATCAACGTAG (SEQ ID NO: 68)GTATCGGCGAACACGAAATCCTCTACTCTTGACAAACTCCCATTCCTACCTC 81 80TCCAAAGTTAGAGGAGAT (SEQ ID NO: 69)TTGCATTACAATGGCCGATCAAGATAAGGACATTCATAATGGAGCTATAGA 79 79ATACAACACCAACGTCGCA (SEQ ID NO: 70)TAATTCTTCCTTGATTCCGTGATTGGATGTCCCTCAGGAGTAGTAGTGTGGA 79 78TGTTGTTGTTAGACACTT (SEQ ID NO: 71)TGGAGGGTCGTAACCGCTATAGATGTGATTCACTCCAACAACTTCCCTATCT 81 78TTAATCCTCTCACTCCAC (SEQ ID NO: 72)TGAATAAATTCGTTGGCGCTGTAGAGATCGGAGTTCCGGATTCGTACTACT 80 80CGTTTACGGGATTTACAGA (SEQ ID NO: 73)GCTAAAGGAGACTCCGGTTTAAACGTCATCGCAATCTTTGATGGGCAAGCG 81 82AGCACATAGATATGCGTTA (SEQ ID NO: 74)AATATTCTCCGGCATGAATGGCGTGGGAATGAATCCGGCTTTGTGTTTATTG 82 81TACATAGACGTTGTCCCG (SEQ ID NO: 75)GAGAACGAGCGGAGCAAGATAGCCTTTAACTGAATCGTCGTCTTATTCCCA 81 79GTACACATCATTCCAAATG (SEQ ID NO: 76)ATATTCTGTACTCAGTGCCTATCCACCTAATAGGGACCTCAGCGACCTGTCC 78 81GTTACATTAATGAAACAT (SEQ ID NO: 77)CATTCCGTAGAATTACTACACCGCGGGATCATTATAACGTCGAAGAGCTTC 79 81AGAGGTAAGTGAAACAAGG (SEQ ID NO: 78)CCCGAAGGCATAATCAACATCCATTGTACATCCCTTGTTATAGCTCCAGGG 81 79CCAGAGATTAAAGGAATAG (SEQ ID NO: 79)CTAGGATGTAACTTGCGTTAGTTGCAGATTCGCTATATTGCTTAAGCTCTGA 79 79GCTCCATGTCCAGTAATT (SEQ ID NO: 80)TTCTCGCAGTTGTAAACTTATAGTGTCGCGCCTAGAAATTCATAGCCACAA 81 78ATTCTCTTTGGGCAGAGAT (SEQ ID NO: 81)TATAGTTACCAAGTACTATGGGTTGGTGGAAGCCGAACGTCTGTCCAAATG 80 80GAGCTATAGTTAAGAGGGA (SEQ ID NO: 82)AGACGCACACCGATAGAGGAGAGATCTTACATACCTGCTAAGGTTGTTAAT 81 79GGCATTGCAGATAGCTTAG (SEQ ID NO: 83)CCAGAAAGGTACAGGGCCAATTAACACGTAATCGGCCTCCAACTCTGCCAT 82 80CTTTAAGCATTCTAAAGCT (SEQ ID NO: 84)AATTCTCCGTCATGTGGTCGTCTGATGCCTAACTTTATCTGCTATCAATGTA 82 79GAGGATCGTGCATTACCG (SEQ ID NO: 85)CGCGGGCTAAGTAGTAGGGTTCTAATGCTACTTTAAATACGCTCACAATCC 80 81AGGCTATATCGCTGTAGCT (SEQ ID NO: 86)TAATCACTGTATTTGTTAATCATGGCTAGGCGGGTCCAATAGGGAAACTGA 79 81TACTAACGTAGGAGCACGC (SEQ ID NO: 87)GTATTCTGGAGAACCTCGTGGCAATGGCAATTCTCCACGAGTGCTAAGATC 82 81TGAGCCGTTTACCAAAGAG (SEQ ID NO: 88)ATAACCTGGTCTCCGGTTGATCGTTTACCTGAAACATGAGATTAGCAACGA 81 82CCCAAACATGCCACTTCAC (SEQ ID NO: 89)CACAACATGCAGCAGGCAAGTAGGGTTTCTGATTATAAGCATCCAGCAATA 81 81AAGCCTCCTTCAAACCAAC (SEQ ID NO: 90)CCCTAACCATGTTCTACGAGCGGTCACAGATTATATTCAACTACAAGTGTA 80 80AATGTACGAGCGCCGAGAT (SEQ ID NO: 91)GAAAGGCATTTGACGGGAGCATTGACGAAGACATACGGTAATTTGTCGTCG 82 81CACGGACAATTAGTGAGTT (SEQ ID NO: 92)TAATACTGGGTCACAAGATTAGATTCCAGCTGTGACGGCGATGAAGTCCGC 78 81GAGGATATGTTTCTATATC (SEQ ID NO: 93)GGTTCATTGTCTCATCGTACGGCTAATGTAGATACGAGGTAGCCGAGTATG 78 82ACACACCACAGCAGTTAAT (SEQ ID NO: 94)TTATGGATTCCGATGATCCTCCGCGTGGTACAAATGTTACCTTGATGCAATA 82 80GTCTCTGTATGCGATCGG (SEQ ID NO: 95)AGCGGTACTAATATGCTATGAGCGAGTTCCCTAACGAGAGATAACGACCCT 80 81CTGTCGTAAGCACTTAAGG (SEQ ID NO: 96)GAGGCATCTCTGCTAACTATATGCTGAACAGCTTTTCCACGATATAGGTAC 80 79ATTGGACGCTTACAGGATA (SEQ ID NO: 97)TTTCGGCCCAACTTATATGCTCTCCGAATCTTGGAGCAGTCATCGTAACCTG 82 80ATAGCAATCTACGTCAAG (SEQ ID NO: 98)ACTGCAGTGAGGGCAACCAATACAAATTAAATCTGCCTCCTATTGGGATAC 79 80CTCCCGTCCATTAAGTTAG (SEQ ID NO: 99)TTGGAGAAACAACCATACAGGTGTCTTTAACTACCTGGAACTCTACCAATT 78 80GGAGCTTTCTTAGCTGTCT (SEQ ID NO: 100)GCTATCAACTTCCCTATCCAAACCGTTGGATGAATTGAAAGCATAGATGTT 80 81CCTTGGAGAGGTTTCCCAG (SEQ ID NO: 101)TGAGGAGTAAGTATACGACGCCTGCACTAGTCACTTGCTGGCTTTGAGCCA 82 81ATAGATGTGTTAATGGCTA (SEQ ID NO: 102)CACAGCCAATCTCTTAGGACAGTACATGGTTAGTAACGTCTGTGGAAGTCA 79 82TGAGCACACGATCTGTAAG (SEQ ID NO: 103)TGAGTATCTACAGGTGTTCTCATGGGATCGTAGTTGGTCTGTCCAACATGAC 79 81GTTATAGGCATAACTCCA (SEQ ID NO: 104)TACCTTAAACTGCGCTGGTAACTTGGATCGTGTAGTCATTGGGAGCAAACC 81 81ATCTGTCTTTCGTATGGAG (SEQ ID NO: 105)GTTAGGTTCAGCCTCATTCCCTAAGAATCCAACTCATAACTCAATCATGCGC 80 81GTCCAGCAAAGACAAATG (SEQ ID NO: 106)ACTGTCTAATACAACCGGATTCTAAGACCACATGGTCTTAGACGCGCGTGC 79 79AATTCTGAACTATATGATT (SEQ ID NO: 107)TGGCTATTGCCGCAGTAGATCAAAGATTGAGAGAGATATAGATTACTCCAT 81 79GATACACCCAAGCCTCGAC (SEQ ID NO: 108)GCAACAAGTGATGCTGACGCAGTTGTTATAGATGGCCTTTGGCTCACGCTA 81 80ATTGAGTTACTGTAGGAAA (SEQ ID NO: 109)GCTATCTCACCAGCTCCTCACCATGACATTTACTCTCCACATTTATCTGCGA 81 80CCTGTTTCGTAAACGATG (SEQ ID NO: 110)

The 70-mer sequence length was selected for optimal hybridization withthe NanoString capture and reporter probes. Other sizes were evaluatedas well. Shortening sequence length tended to improve signal but reducehybridization capability. For example, although 50-mer sequences gaverelatively higher signals when compared to controls, 30-mer sequencesdid not reliably hybridize. Thus, 70-mer sequences were selected forreliable hybridization. However, sequences that are longer or shorterthan 70 nucleotides can also be used in the methods described herein.

Antibodies (e.g., listed in Table 1) can be purchased from commercialsources, and were initially purified from BSA and/or other contaminantswith either a Zeba spin column or centrifugal filter. Antibodies werethen incubated with photocleavable bifunctional linker in PBS(containing 5% N,N′-dimethylformamide and 10% 0.1 M NaHCO₃) at roomtemperature for 1.5 hours. Afterward, excess reagents were removed frommaleimide-activated antibodies with a Zeba spin column [7000 molecularweight cutoff (MWCO), eluent: PBS].

Thiol-modified DNA oligos (from Integrated DNA Technologies) werereduced with dithiothreitol (DTT; 100 mM) in PBS (1 mM EDTA, pH 8.0) for2 hours at room temperature. The reduced DNA oligos were then purifiedwith NAP-5 column (GE Healthcare), with deionized water as the eluent.The fractions containing DTT (determined with the microBCA assay) werediscarded. The remaining reduced DNA fractions were pooled andconcentrated with a 3000 MWCO Amicon filter (Millipore).

The maleimide-activated antibodies were incubated with the reduced DNAoligos in PBS solution. In a typical conjugation process, 15-fold molarexcess of DNA oligos was incubated with maleimide-activated antibodies.The conjugation reaction was allowed to proceed for 12 hours at 4° C.DNA barcode-antibody conjugates were purified with a Millipore 100K MWCOcentrifugal filter followed by three washes with PBS. After theantibodies were mixed, a final purification of excess DNA was conductedwith protein A/G-coated magnetic beads (Pierce/Thermo Scientific). Thecommercial protocol from Thermo for magnetic separation was onlyslightly modified to use a tris-buffered saline (TBS)/0.1% Tween washbuffer and a Gentle Ag/Ab Elution Buffer (Thermo Scientific). Threeelutions were performed for 20 min each. Solvent antibody was exchangedinto pure TBS with a Zeba desalting column (7000 MWCO).

Antibody characterization. Antibodies were aliquoted and stored atconcentrations of 0.25 mg/ml in PBS with BSA (0.15 mg/ml) at −20° C.,with adequate usage for at least twelve experimental runs (the number ofruns on each NanoString cartridge) to avoid freeze-thaw cycles. Variousother storage methods were tested, including glycerol or 4° C. storage,but aliquoting and freezing showed the most consistent, high-fidelitystorage for up to 9 months. Antibody concentrations were determined viamicroBCA assay (Thermo Scientific). DNA concentrations were alsoindependently determined using the Qubit ssDNA kit (Invitrogen) toquantify the relative number of DNA per antibody. To achieve relativeDNA/Ab measurements with higher sensitivity across the cohort ofantibodies, in some embodiments, the NanoString platform was used to addantibody cocktails under two conditions: (1) “Control”: antibodies wereadded in their native forms with DNA still attached, and (2) “ReleasedDNA”: antibodies were treated with proteinase K and photocleaved. Underthe control condition, the DNA was still attached to the antibody andthus could not simultaneously bind to the NanoString assay's reporterand capture probe. The difference in DNA readings between these twomeasurements thus revealed the relative number of DNA per antibody. Thisdifference was divided by the isotype control measurement to account forpossible inherent experimental error in protein concentration and/orantibody isolation (see FIG. 6 for relative number of DNA:Ab ratio).Antibodies were rigorously tested and validated prior to use. Of 110antibodies, 88 were selected for the final panel and all had beenpreviously validated from specific vendors (primarily Cell SignalingTechnologies, BioLegend; Table 1). Antibodies that did not work with DNAconjugates did not work in their native state either and were excluded;DNA conjugated antibodies worked as well as the parent antibody (FIGS.7A-7B).

Antibody staining and DNA collection for protein profiling. Prior tocell staining, antibodies were pooled into a cocktail with TBS, 0.1%Tween, and 0.2 mg/ml cysteine (to avoid DNA cross-reaction with otherantibodies). Tubes were coated with serum blocking buffer overnight toprevent samples from non-specifically binding to tube walls. Cells werethen incubated for a minimum of one hour with a blocking buffer at 37°C.: 10% v/v Rabbit serum (Jackson Immuno Research Labs, 011-000-120), 2%BSA, 1 mg/ml SS salmon sperm DNA (Sigma Aldrich, D7656), 0.2 mg/mlcysteine (Sigma Aldrich), 20× Perm (BD Bioscience) or 0.1% Tween 20(Sigma Aldrich)—all in PBS to minimize non-specific antibody or DNAbinding. The antibody cocktail was then added to the fixed andpermeabilized cells and incubated for one hour at RT with intermittentmixing.

After incubation, the cells were washed with PW+ with 0.05 mg/ml of DSsheared salmon sperm DNA (Life Technology, AM9680). Either two 15-mlwashes in 15-ml tubes or four 1.5-ml washes in 1.7-ml microcentrifugetubes were performed. Blocking and wash steps were desired for achievinglow background even with femtomolar detection. All washes were performedon ice. Labeled cells could then be counted and selected forlysis/proteinase K/photocleavage to release the DNA. Lysis buffer wasused on 10 μl of cells (with up to 50,000 cells), 34.2 μl of ATL lysisbuffer (Qiagen) and 5.8 μl of Proteinase K (Qiagen). This reactionproceeded at 56° C. for a minimum of 30 min. Photocleavage was thenperformed using long UV wavelength light (model) for 15 min. Thisresulted in a cell-lysis mix with released DNA. Samples were spun downat 14,000×g for 10 min. Supernatant was collected, and serial dilutionswere performed in nuclease-free water (Invitrogen, AM9937) to collectDNA equivalent to 50-100 cells to avoid saturating the read-outcartridge (Nanostring). This amount resulted in cartridge bindingdensities within the linear range of quantitation. Binding densities inthe lower range (0.05-0.2) were still linear and gave consistent proteinprofiles comparable to those in the higher range (1.5-2.5). At lowerbinding densities (for example single cells), the majority of markerscould be measured, with the exception of low expression markers withweaker antibodies (pJAK2, pChk2).

Immunofluorescence. Immunofluorescence provided an independent measureand validated marker changes from paclitaxel (Taxol) treatment (FIGS.16A-16B). HT1080 cells were seeded at 4,000 cells per well in 96 wellplates (Grenier), which were compatible with high resolution plates, andgrown for 24h in DMEM media before either being treated with Paclitaxelat 100 nM or kept in control media. After 24 h, cells were fixed for 15min at RT, then gently washed on a rocker with PBS/0.1% Tween for 5 min,repeated 3 times. All subsequent washes were also performed with thisbuffer, time duration, and repetition protocol.

Cells were then permeabilized with ice cold 90% methanol for 20 min.After washing, cells were blocked for 1 h at room temperature withblocking buffer (Odyssey). Primary antibodies (all from Cell Signaling;see Table 1) were then added in blocking buffer at prescribed dilutions,sealed, covered in foil and incubated overnight. The next day, afterwashing, anti-rabbit-FITC secondary antibodies, 1:500 Hoechst and 1:200whole-cell stain blue (Cellomics) were added (all primary antibodieswere rabbit IgGs) at 2 mg/ml and incubated for 2 h. Final wash stepswere performed in PBS only, and the cells were subsequently imaged at20× using an Olympus microscope (BX63) with a Delta Vision chamber andsoftware. All images were taken in biological triplicate. Fluorescenceintensity for each cell was determined using CellProfiler, which usedHoechst and whole-cell stain to delineate cell boundaries and sizeconstraints to discount debris. Additional in-house MATLAB (Mathworks)code was then used to calculate marker signals for each condition andcalculate the changes between treated and untreated cells.

Immunoblotting and dot blotting. OVCAR3, SKOV3, CAOV3, A2780, andOVCAR429 cell lines were plated in 6-well dishes and grown for 72 hprior to lysis for Western blot analysis. Cells were washed twice withice-cold PBS, scraped into 200 μl per dish of radio immunoprecipitationassay buffer (RIPA buffer) (Cell Signaling Technology), containing HALTprotease and phosphatase inhibitor cocktail (Pierce), and transferred to2 ml microcentrifuge tubes. Lysates was passed through a 23-g syringe 5times, and then incubated 5 min on ice with vortexing every min. Lysateswere centrifuged 15 min at 14,000×g (4° C.). Supernatant was transferredto a new microcentrifuge tube and total protein was measured using theBCA assay. Equal total protein was prepared, boiled, and loaded on aNovex NuPAGE 4-12% Bis-Tris gel and then transferred to nitrocellulose.

Membranes were blocked for 1 h at room temperature in SuperBlock T20(TBS) buffer (Pierce) and then washed briefly in tris-buffered saline(TBS) with 0.1% Tween-20 (TBST). Membranes were then incubated overnightat 4° C. with rocking in p53 (1:1000, Cell Signaling), DNA-conjugatedp53 (1:1000), pS6RP (1:1000, Cell Signaling), or DNA-conjugated pS6RP(1:1000) primary antibodies diluted in TBST with 10% SuperBlock.Membranes were washed three times, 5 min each in TBST and then incubated1 h at room temperature in goat a-rabbit HRP conjugated secondaryantibody diluted 1:1000 in TBST with 10% SuperBlock. Following washing,signal was detected using SuperSignal West Pico chemiluminescentsubstrate (Pierce). For the Ki67 antibodies, lug cell lysates (prior todenaturing) from above were loaded onto nitrocellulose a Bio-Dotmicrofiltration apparatus (Bio-Rad). Blots were then processed as above,using a Ki67 or DNA-conjugated Ki67 antibody (1:1000, BD Biosciences)and an α-mouse HRP conjugated secondary antibody diluted as above. Dotblots were detected as above.

Single-cell isolation and processing. After antibody staining, singlecells were picked with a micromanipulator. Cells were stained withHoechst 3342 (Molecular Probes), added to an open 10-cm dish, and imagedwith a TE2000 microscope (Nikon). Single cells were placed directly intoa PCR tube. Five microliters of lysis buffer/proteinase K was added (4.5μl of ATL buffer and 0.5 μl of proteinase K). Lysis/enzymatic cleavageproceeded for 30 min at 56° C. before photocleavage for 15 min. Reporterand capture probes (NanoString Technologies) were then directly added tothis tube according to the manufacturer's recommendations.

Data analysis: calculating proteomic expression profiles. Proteinexpression profiles were extracted from raw data as follows. First, rawDNA counts were normalized via the mean of the internal NanoStringpositive controls, which account for hybridization efficiency. Thesecounts were then converted to antibody expression values using therelative DNA/antibody counts. Next, average background signal fromcontrol IgG was subtracted. Last, housekeeping genes were used fornormalization that accounted for cell number variations. Signals werenormalized via a house-keeping protein, e.g., β-tubulin. For the taxoltreatments, signals were normalized via the geometric mean of histoneH3, GAPDH, and actin rather than tubulin, because tubulin is a primarytarget of taxol. Data were transformed into log 2 scale as denoted incaptions.

Data analysis: clustering. Heat maps and clustergrams were plotted usingMATLAB with a matrix input of marker expression values that werecalculated as detailed above. All shown clustergrams were performed as aweighted linkage and were clustered using correlation values as adistance metric. Some clustergrams were normalized by row, as specifiedin captions, to highlight marker differences among different patients.If a marker was not detectable in one of the patients, it was removedfrom the matrix or heat map and is not displayed.

Statistical analysis. Raw data from NanoString DNA counts werenormalized by first using the nSolver analysis software to account forhybridization differences on the cartridge. Only positive controls A toD on the NanoString software were used in normalization. DNA counts werewithin the linear range of detection and met all other criteria forinclusion as determined by the nSolver software (maximum fields of view,image quality, etc.). After determining an expression value by takinginto account nonspecific IgG binding and housekeeping genes (cellcount), data were log 2-transformed.

Correlation between single-cell analysis and bulk measurement wascalculated in GraphPad Prism. Spearman r values were calculated withoutassuming a normal, consistent distribution. Two-sided P values werecalculated, where significant markers were identified by comparing twogroups (for example, treated versus untreated) in Prism and performingpairwise t tests with an FDR of 0.2 for multiple test correction error.Significant marker changes and their P values between gefitinib-treatedand untreated A431 single cells are shown in Table 3 below. For heatmaps, if any samples had markers below threshold, the entire marker rowwas removed (no imputed data values were used). To identifydifferentiating markers between responders and nonresponders, amulticlass sequential forward selection-ranking algorithm was used. Thepatients were classified as responders or nonresponders based on knowndata. Class separability was measured by the Bhattacharya distance.

TABLE 3 Significant markers between A431 single cells with or withoutgefitinib treatment. Six markers out of 49 markers showed significantdifference between gefitinib-treated vs. untreated A431 single cells andthe average expression values as calculated via Nanostring profiling foreach cohort. Marker significance was determined by pairwise t-testingand corrected for multiple testing errors by using a false discoveryrate of 0.2. Proteins P Untreated Treated Phospho-S6RP 0.0067212 1171.358.4 Phospho-histone H3 0.0091305 4920.6 982.0 Ku80 0.0098001 770.2120.6 FGFR4 0.0106319 914.9 114.1 CD56 0.0117795 1906.5 334.4Dimethyl-histone H3 (Lys36) 0.0119939 695.7 86.9

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What is claimed is:
 1. A method for detecting a plurality of targetmolecules in a sample comprising: a. contacting a sample with acomposition comprising a plurality of target probes, wherein each targetprobe in the plurality comprises: i. a target-binding molecule thatspecifically binds to a distinct target molecule in the sample; ii. anidentification nucleotide sequence that identifies the target-bindingmolecule; and iii. a cleavable linker between the target-bindingmolecule and the identification nucleotide sequence; b. separatingunbound target probes from a plurality of complexes in the sample, eachcomplex having a target molecule and a single target probe boundthereto, wherein the complex does not have a second target probe bindingto a different region of the target molecule; c. releasing theidentification nucleotide sequences from the plurality of complexes; d.coupling the released identification nucleotide sequences from thereleasing step (c) to a detection composition comprising a plurality ofreporter probes, wherein each reporter probe in the plurality comprisesa detectable label that identifies the reporter probe, wherein the labelcreates a unique distinguishable signal for each reporter probe; and e.detecting signals from the released identification nucleotide sequencesbased on a non-gel electrophoresis method, wherein the signals aredistinguishable for the identification nucleotide sequences, therebyidentifying the corresponding target-binding molecules and detecting aplurality of different target molecules in the sample.
 2. The method ofclaim 1, wherein the composition further comprises a plurality ofcontrol probes, wherein each control probe in the plurality comprises acontrol-binding molecule that specifically binds to one control moleculein the sample; an identification control sequence that identifies thecontrol-binding molecule; and a cleavable linker between thecontrol-binding molecule and the identification control sequence, andwherein the method, further comprises quantifying the signals bynormalizing the signals associated with the target probes by the signalsassociated with the control probes.
 3. The method of claim 1, whereinthe detecting step (e) comprises no amplification of the releasedidentification nucleotide sequences.
 4. The method of claim 1, whereinthe target-binding molecule is an antibody.
 5. The method of claim 1,wherein the target-binding molecule is a nucleic acid.
 6. The method ofclaim 1, wherein the cleavable linker is a cleavable non-hybridizablelinker.
 7. The method of claim 6, wherein the cleavable,non-hybridizable linker is sensitive to an enzyme, pH, temperature,light, shear stress, sonication, a chemical agent, or any combinationthereof.
 8. The method of claim 6, wherein the cleavable,non-hybridizable linker comprises a photocleavable linker.
 9. The methodof claim 1, wherein the releasing of the identification nucleotidesequences from the bound target probes comprises exposing the boundtarget probes to ultraviolet light.
 10. The method of claim 1, whereinthe detection composition further comprises a plurality of captureprobes, wherein each capture probe comprises an affinity tag.
 11. Themethod of claim 10, wherein the affinity tag of the capture probepermits immobilization of the released identification nucleotidesequences onto a solid substrate, upon coupling to the detectioncomposition.
 12. A kit for multiplexed detection of a plurality ofdifferent target molecules from a sample comprising: a. a plurality oftarget probes, wherein each target probe in the plurality comprises: i.a target-binding molecule that specifically binds to a distinct targetmolecule in the sample; ii. an identification nucleotide sequence thatidentifies the target-binding molecule; and iii. a cleavable,non-hybridizable linker between the target-binding molecule and theidentification nucleotide sequence; b. a plurality of reporter probes,wherein each reporter probe comprises: i. a first target probe-specificregion that is capable of binding a first portion of the identificationnucleotide sequence; and ii. a detectable label that identifies thereporter probe; and c. a plurality of capture probes, wherein eachcapture comprises: i. a second target probe-specific region that iscapable of binding a second portion of the identification nucleotidesequence; and ii. an affinity tag for immobilization of theidentification nucleotide sequence to a solid substrate surface.
 13. Thekit of claim 12, wherein the target-binding molecule is an antibody. 14.The kit of claim 12, wherein the target-binding molecule is a nucleicacid.